Maize plants with improved disease resistance

ABSTRACT

The present invention is in the field of plant breeding and disease resistance. More specifically, the invention includes a method for breeding corn plants containing one or more markers that are associated with resistance to fungi. The invention further includes germplasm and the use of germplasm containing at least one marker associated with resistance to Fusarium stalk rot (FSR) infection for introgression into elite germplasm in a breeding program, thus producing novel FSR resistant germplasm.

REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/331,918, filed May 4, 2016, which is herein incorporated by referencein its entirety.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named “MONS390US_ST25.txt” whichis 80,783 bytes (measured in MS-Windows®) and created on May 1, 2017,and comprises 230 sequences, and is incorporated herein by reference inits entirety.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and morespecifically to methods and compositions for producing corn plantsexhibiting improved disease resistance.

BACKGROUND

Stalk rot infection reduces the efficiency of carbohydrate transportfrom the stalk up to the ears during grainfill, which reduces cropyield. A corn plant will die altogether if infection advances to thepoint that the pith pulls away from the outer rind of the stalk, whichcan eventually result in a stalk consisting of little more than a hollowtube that is no longer able transport water and nutrients to the rest ofthe plant. Furthermore, a stalk weakened by infection is more likely tocollapse at one or more points along its length (lodging), whichtypically results in a plant that yields no harvestable grain. Stalkrots typically reduce yields up to 5% in almost any field where corn iscultivated. In years with particularly bad infection rates, yield lossesreach 10-20%, and in some locations when infection is particularlyacute, 100% yield loss can occur.

One of the most common forms of stalk rot is Fusarium stalk rot (FSR),caused by several species of fungi, including Fusarium verticilliodes,Fusarium proliferatum, and Fusarium subglutinans. FSR infection ischaracterized by rotting roots, crown, and lower internodes that beginsshortly after pollination and progresses as the plant matures.Eventually the pith will disintegrate resulting in weak, spongy stalksthat are prone to lodging.

Due to the lack of fungicides and or other chemical controls for FSR,growers are faced with limited options for managing the disease. Sincethe most effective approach is to select hybrids that are intrinsicallyresistant, what is needed are methods of identifying genetic sources ofFSR resistance and more effective methods of introgressing those geneticelements into commercial lines to provide new hybrids with improvedgenetic resistance to FSR infection.

SUMMARY OF THE INVENTION

Identifying and selecting plants that exhibit resistance to Fusariumstalk rot (FSR) using marker-assisted selection (MAS) provides aneffective and efficient method of improving the survivability of corn toFSR infection. This invention provides marker loci and quantitativetrait loci (QTL) chromosome intervals that demonstrate significantco-segregation with FSR resistance. These markers, or additional locilinked to these markers, can be used in MAS breeding programs to produceplants with improved FSR resistance.

Marker loci and quantitative trait loci (QTL) chromosome intervals thatdemonstrate significant co-segregation with FSR resistance are provided.These markers, or additional loci linked to these markers, can be usedin MAS breeding programs to produce plants with improved FSR resistance.

The FSR_2.01, FSR_5.01, FSR_6.01 and FSR_7.01 loci correspond to QTLdiscovered on chromosomes 2, 5, 6, and 7, respectively, of the corngenome. These loci contain genotypes closely linked to FSR resistanceEmbodiments of this invention include methods of detecting genotypeswithin and/or linked to FSR_2.01, FSR_5.01, FSR_6.01 or FSR_7.01 tocreate disease resistant corn lines. Provided herein are examples ofmarkers that are useful for detecting the presence or absence of diseaseresistance alleles linked to FSR_2.01, FSR_5.01, FSR_6.01 or FSR_7.01 aspart of a MAS breeding program to produce plants with improvedresistance to FSR infection.

Embodiments of this invention include identifying one or more cornplants with FSR resistance, improved resistance, or susceptibility toFSR infection by using a marker within the FSR_2.01, FSR_5.01, FSR_6.01or FSR_7.01 chromosome intervals, or a marker closely linked toFSR_2.01, FSR_5.01, FSR_6.01 or FSR_7.01. As used herein, “closelylinked” means that the marker or locus is within about 20 cM, preferablywithin about 15 cM, more preferably within about 10 cM, even morepreferably within about 5 cM, even more preferably within about 1 cM,even more preferably about 0.5 cM, and even more preferably less than0.5 cM of the identified FSR locus.

The location in the maize genome of FSR_2.01, FSR_5.01, FSR_6.01 andFSR_7.01, and chromosome intervals and sub-intervals containing markersclosely linked to FSR_2.01, FSR_5.01, FSR_6.01 and FSR_7.01, arereferenced herein to a public maize genome map (IBM2 2008 Neighbors).Genomic markers such as npi610 and gpm365a can be used to define theflanks of the FSR_2.01 chromosome interval, which includes markersclosely linked to FSR_2.01. Genomic markers such as hmg2 and agrp90 canbe used to define the flanks of the FSR_5.01 chromosome interval, whichincludes markers closely linked to FSR_5.01. Genomic markers such asgpm674c and TIDP5534 can be used to define the flanks of the FSR_6.01chromosome interval, which includes markers closely linked to FSR_6.01.Genomic markers such as TIDP2919 and IDP5002 can be used to define theflanks of the FSR_7.01 chromosome interval, which includes markersclosely linked to FSR_7.01. Other genomic markers may be used to definechromosome sub-intervals linked to FSR-FSR_2.01, FSR_5.01, FSR_6.01 orFSR_7.01.

Embodiments of this invention include methods of creating a populationof corn plants with enhanced FSR resistance by providing a firstpopulation of corn plants, detecting the presence of a genetic markerthat is genetically linked to FSR_2.01 by 20 cM or less in the firstpopulation, selecting one or more corn plants containing said markerfrom the first population of corn plants, and producing a population ofoffspring from at least one of said selected corn plants. In otherembodiments, the genetic marker detected is genetically linked toFSR_2.01 by less than 15 cM, 10 cM, 5 cM, 1 cM, or 0.5 cM of FSR_2.01.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker within theFSR_2.01 chromosome interval, selecting one or more corn plantscontaining said marker from the first population of corn plants, andproducing a population of offspring from at least one of said selectedcorn plants. The FSR_2.01 chromosome interval includes any markerflanked by npi610 and gpm365a, including SEQ ID NOs: 1-3. Sub-intervalsof the FSR_2.01 chromosome interval are also useful for this invention,and include any interval wherein one or both borders of the sub-intervalare between npi610 and gpm365a. In one embodiment, an FSR_2.01sub-interval is flanked by, and includes, SEQ ID NO: 1 and SEQ ID NO: 3.All manner of chromosome interval lengths between, and including, npi610and gpm365a can be used in conjunction with this invention.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker that isselected from the group consisting of SEQ ID NOs: 1-3 in the firstpopulation, selecting one or more corn plants containing said markerfrom the first population of corn plants, and producing a population ofoffspring from at least one of said selected corn plants.

Embodiments of this invention further include methods of creating apopulation of corn plants with enhanced FSR resistance by providing afirst population of corn plants, detecting the presence of a geneticmarker that is genetically linked to FSR_5.01 by 20 cM or less in thefirst population, selecting one or more corn plants containing saidmarker from the first population of corn plants, and producing apopulation of offspring from at least one of said selected corn plants.In other embodiments, the genetic marker detected is genetically linkedto FSR_5.01 by less than 15 cM, 10 cM, 5 cM, 1 cM, or 0.5 cM ofFSR_5.01.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker within theFSR_5.01 chromosome interval, selecting one or more corn plantscontaining said marker from the first population of corn plants, andproducing a population of offspring from at least one of said selectedcorn plants. The FSR_5.01 chromosome interval includes any markerflanked by hmg2 and agrp90, including SEQ ID NOs: 4-7. Sub-intervals ofthe FSR_5.01 chromosome interval are also useful for this invention, andinclude any interval wherein one or both borders of the sub-interval arebetween hmg2 and agrp90. In one embodiment, an FSR_5.01 sub-interval isflanked by, and includes, SEQ ID NO: 4 and SEQ ID NO: 7. All manner ofchromosome interval lengths between, and including, hmg2 and agrp90 canbe used in conjunction with this invention.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker that isselected from the group consisting of SEQ ID NOs: 4-7 in the firstpopulation, selecting one or more corn plants containing said markerfrom the first population of corn plants, and producing a population ofoffspring from at least one of said selected corn plants.

Embodiments of this invention further include methods of creating apopulation of corn plants with enhanced FSR resistance by providing afirst population of corn plants, detecting the presence of a geneticmarker that is genetically linked to FSR_6.01 by 20 cM or less in thefirst population, selecting one or more corn plants containing saidmarker from the first population of corn plants, and producing apopulation of offspring from at least one of said selected corn plants.In other embodiments, the genetic marker detected is genetically linkedto FSR_6.01 by less than 15 cM, 10 cM, 5 cM, 1 cM, or 0.5 cM ofFSR_6.01.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker within theFSR_6.01 chromosome interval, selecting one or more corn plantscontaining said marker from the first population of corn plants, andproducing a population of offspring from at least one of said selectedcorn plants. The FSR_6.01 chromosome interval includes any markerflanked by gpm674c and TIDP5534, including SEQ ID NOs: 8-23.Sub-intervals of the FSR_6.01 chromosome interval are also useful forthis invention, and include any interval wherein one or both borders ofthe sub-interval are between gpm674c and TIDP5534. In one embodiment, anFSR_6.01 sub-interval is flanked by, and includes, SEQ ID NO: 8 and SEQID NO: 23. All manner of chromosome interval lengths between, andincluding, gpm674c and TIDP5534 can be used in conjunction with thisinvention.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker that isselected from the group consisting of SEQ ID NOs: 8-23 in the firstpopulation, selecting one or more corn plants containing said markerfrom the first population of corn plants, and producing a population ofoffspring from at least one of said selected corn plants.

Embodiments of this invention further include methods of creating apopulation of corn plants with enhanced FSR resistance by providing afirst population of corn plants, detecting the presence of a geneticmarker that is genetically linked to FSR_7.01 by 20 cM or less in thefirst population, selecting one or more corn plants containing saidmarker from the first population of corn plants, and producing apopulation of offspring from at least one of said selected corn plants.In other embodiments, the genetic marker detected is genetically linkedto FSR_7.01 by less than 15 cM, 10 cM, 5 cM, 1 cM, or 0.5 cM ofFSR_7.01.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker within theFSR_7.01 chromosome interval, selecting one or more corn plantscontaining said marker from the first population of corn plants, andproducing a population of offspring from at least one of said selectedcorn plants. The FSR_7.01 chromosome interval includes any markerflanked by TIDP2919 and IDP5002, including SEQ ID NOs: 24-27.Sub-intervals of the FSR_7.01 chromosome interval are also useful forthis invention, and include any interval wherein one or both borders ofthe sub-interval are between TIDP2919 and IDP5002. In one embodiment, anFSR_7.01 sub-interval is flanked by, and includes, SEQ ID NO: 24 and SEQID NO: 27. All manner of chromosome interval lengths between, andincluding, TIDP2919 and IDP5002 can be used in conjunction with thisinvention.

In another embodiment of this invention, a population of corn plantswith enhanced FSR resistance is created by providing a first populationof corn plants, detecting the presence of a genetic marker that isselected from the group consisting of SEQ ID NOs: 24-27 in the firstpopulation, selecting one or more corn plants containing said markerfrom the first population of corn plants, and producing a population ofoffspring from at least one of said selected corn plants.

In one aspect, the present invention provides a method of obtaining acorn plant with enhanced Fusarium stalk rot resistance comprising: a)providing a population of corn plants; b) detecting in said plants thepresence of a Fusarium stalk rot resistance allele at a polymorphiclocus genetically linked to a chromosomal segment; and c) selecting fromsaid population at least a first plant comprising said allele andenhanced Fusarium stalk rot resistance compared to a plant lacking saidallele. In some embodiments, said segment is flanked by marker locinpi610 and gpm365a on chromosome 2, or flanked by marker loci hmg2 andagrp90 on chromosome 5, or flanked by marker loci gpm674c and TIDP5534on chromosome 6, or flanked by marker loci TIDP2919 and IDP5002 onchromosome 7. In further embodiments, said segment is flanked by markerloci SEQ ID NO: 1 and SEQ ID NO: 3 on chromosome 2, or by marker lociSEQ ID NO: 4 and SEQ ID NO: 7 on chromosome 5, or by marker loci SEQ IDNO: 8 and SEQ ID NO: 23 on chromosome 6, for example by marker loci SEQID NO: 141 and SEQ ID NO: 143 on chromosome 6, or by marker loci SEQ IDNO: 24 and SEQ ID NO: 27 on chromosome 7. In other embodiments, saidpolymorphic locus comprises a nucleic acid sequence selected from thegroup consisting of SEQ ID NOs: 1-27 and 136-154. In some embodiments,said method further comprises selecting from said population at leasttwo plants, thereby forming a population of corn plants comprising saidallele and enhanced Fusarium stalk rot resistance compared to a plantlacking said allele. In certain embodiments, said Fusarium stalk rotresistance allele was introgressed into said population of corn plantsfrom a starting plant or population of corn plants containing saidallele. In other embodiments, said method further comprises producing aprogeny plant with Fusarium stalk rot resistance from said first plant.In some embodiments, producing the progeny plant comprisesmarker-assisted selection for Fusarium stalk rot resistance. In furtherembodiments, said progeny plant is an F2-F6 progeny plant. In yetfurther embodiments, producing said progeny plant comprisesbackcrossing.

In another aspect, the invention provides a method of producing a cornplant with enhanced Fusarium stalk rot resistance comprising: a)crossing a first corn plant comprising a Fusarium stalk rot resistanceallele with a second corn plant of a different genotype to produce oneor more progeny plants; and b) selecting a progeny plant based on thepresence of said allele at a polymorphic locus genetically linked to achromosomal segment; wherein said allele confers enhanced resistance toFusarium stalk rot compared to a plant lacking said allele. In someembodiments, said segment is flanked by marker loci npi610 and gpm365aon chromosome 2, or flanked by marker loci hmg2 and agrp90 on chromosome5, or flanked by marker loci gpm674c and TIDP5534 on chromosome 6, orflanked by marker loci TIDP2919 and IDP5002 on chromosome 7. In furtherembodiments, said segment is flanked by marker loci SEQ ID NO: 1 and SEQID NO: 3 on chromosome 2, or by marker loci SEQ ID NO: 4 and SEQ ID NO:7 on chromosome 5, or by marker loci SEQ ID NO: 8 and SEQ ID NO: 23 onchromosome 6, for example by marker loci SEQ ID NO: 141 and SEQ ID NO:143 on chromosome 6, or by marker loci SEQ ID NO: 24 and SEQ ID NO: 27on chromosome 7. In some embodiments, said polymorphic locus comprises anucleic acid sequence selected from the group consisting of SEQ ID NO:1-27 and 136-154. In certain embodiments, said progeny plant is an F2-F6progeny plant. In other embodiments, producing said progeny plantcomprises backcrossing. In some embodiments, backcrossing comprises from2-7 generations of backcrosses. In further embodiments, backcrossingcomprises marker-assisted selection in at least two generations. In yetfurther embodiments, backcrossing comprises marker-assisted selection inall generations. In some embodiments, said first corn plant is an inbredor a hybrid. In other embodiments, said second corn plant is anagronomically elite corn plant. In further embodiments, saidagronomically elite corn plant is an inbred or a hybrid.

DETAILED DESCRIPTION OF THE INVENTION I. Chromosome Intervals

The term “chromosome interval” designates a contiguous linear span ofgenomic DNA that resides in planta on a single chromosome. The term alsodesignates any and all genomic intervals defined by any of the markersset forth in this invention. The genetic elements located on a singlechromosome interval are physically linked and the size of a chromosomeinterval is not particularly limited. In some aspects, the geneticelements located within a single chromosome interval are geneticallylinked, typically with a genetic recombination distance of, for example,less than or equal to 20 cM, or alternatively, less than or equal to 10cM. That is, two genetic elements within a single chromosome intervalundergo meiotic recombination at a frequency of less than or equal to20% or 10%, respectively.

The boundaries of a chromosome interval can be defined by geneticrecombination distance or by markers. In one embodiment, the boundariesof a chromosome interval comprise markers. In another embodiment, theboundaries of a chromosome interval comprise markers that will be linkedto the gene controlling the trait of interest, i.e., any marker thatlies within a given interval, including the terminal markers thatdefining the boundaries of the interval, and that can be used as amarker for the presents or absence of disease resistance. In oneembodiment, the intervals described herein encompass marker clustersthat co-segregate with disease resistance. The clustering of markersoccurs in relatively small domains on the chromosomes, indicating thepresence of a genetic locus controlling the trait of interest in thosechromosome regions. The interval encompasses markers that map within theinterval as well as the markers that define the terminal.

An interval described by the terminal markers that define the endpointsof the interval will include the terminal markers and any markerlocalizing within that chromosome domain, whether those markers arecurrently known or unknown. Although it is anticipated that one skilledin the art may describe additional polymorphic sites at marker loci inand around the markers identified herein, any marker within thechromosome intervals described herein that are associated with diseaseresistance fall within the scope of this claimed invention.

“Quantitative trait loci” or a “quantitative trait locus” (QTL) is agenetic domain that effects a phenotype that can be described inquantitative terms and can be assigned a “phenotypic value” whichcorresponds to a quantitative value for the phenotypic trait. A QTL canact through a single gene mechanism or by a polygenic mechanism. In someaspects, the invention provides QTL chromosome intervals, where a QTL(or multiple QTLs) that segregates with disease resistance is containedin those intervals. In one embodiment of this invention, the boundariesof chromosome intervals are drawn to encompass markers that will belinked to one or more QTL. In other words, the chromosome interval isdrawn such that any marker that lies within that interval (including theterminal markers that define the boundaries of the interval) isgenetically linked to the QTL. Each interval comprises at least one QTL,and furthermore, may indeed comprise more than one QTL. Close proximityof multiple QTL in the same interval may obfuscate the correlation of aparticular marker with a particular QTL, as one marker may demonstratelinkage to more than one QTL. Conversely, e.g., if two markers in closeproximity show co-segregation with the desired phenotypic trait, it issometimes unclear if each of those markers identifying the same QTL ortwo different QTL. Regardless, knowledge of how many QTL are in aparticular interval is not necessary to make or practice the invention.

FSR_2.01, FSR_5.01, FSR_6.01, and FSR_7.01 Chromosome Intervals

In one embodiment, the present invention provides a plant comprising anucleic acid molecule selected from the group consisting of SEQ ID NO:1-27 and 136-154, fragments thereof, and complements of both. In anotherembodiment, the present invention also provides a plant comprising thealleles of the FSR_2.01, FSR_5.01, FSR_6.01, or FSR_7.01 chromosomeintervals, or fragments and complements thereof, as well as any plantcomprising any combination of one or more disease resistance loci linkedto at least one marker selected from the group consisting of SEQ ID NOs:1-27 and 136-154. Such alleles may be homozygous or heterozygous.

The locations in the maize genome of FSR_2.01, FSR_5.01, FSR_6.01, orFSR_7.01 and the chromosome intervals comprising markers closely linkedto it are disclosed in Table 6. Genetic map loci are represented in cM,with position zero being the first (most distal) marker known at thebeginning of the chromosome on both Monsanto's internal consensusgenetic map and the Neighbors 2008 maize genomic map, which is freelyavailable to the public from the Maize GDB website and commonly used bythose skilled in the art.

For example, the FSR_2.01 chromosome interval comprises SEQ ID NOs: 1-3and is flanked by the markers npi610 and gpm365a. This chromosomeinterval encompasses a marker cluster that co-segregates with FSRresistance in the populations studied at a p-value ≤0.001.

Similarly, the FSR_5.01 chromosome interval contains SEQ ID NOs: 4-7 andis flanked by the markers hmg2 and agrp90. This chromosome intervalencompasses a marker cluster that co-segregates with FSR resistance inthe populations studied at a p-value ≤0.001.

The FSR_6.01 chromosome interval contains SEQ ID NOs: 8-23 and 136-154and is flanked by the markers gpm674c and TIDP5534. This chromosomeinterval encompasses a marker cluster that co-segregates with FSRresistance in the populations studied at a p-value ≤0.001. The FSR_6.01interval provided herein comprises a chromosomal interval flanked bymarker loci SEQ ID NOs: 141 and SEQ ID NO: 143 and comprising markerlocus SEQ ID NO: 142.

The FSR_7.01 chromosome interval contains SEQ ID NOs: 24-27 and isflanked by the markers TIDP2919 and IDP5002. This chromosome intervalencompasses a marker cluster that co-segregates with FSR resistance inthe populations studied at a p-value ≤0.001.

Thus, one skilled in the art can use this invention to improve theefficiency of breeding for improved disease resistance in maize byassociating disease resistance phenotypes with genotypes at previouslyunknown disease resistance loci in the maize genome. Disclosed hereinare chromosome intervals that comprise alleles responsible forphenotypic differences between disease resistant and disease susceptiblecorn lines. Each chromosome interval is characterized by the genomicregions flanked by and including the markers npi610 and gpm365a onchromosome 2; or hmg2 and agrp90 on chromosome 5; or gpm674c andTIDP5534 on chromosome 6; or TIDP2919 and IDP5002 on chromosome 7, andcomprise markers within or closely linked to (within 20 cM of) FSR_2.01,FSR_5.01, FSR_6.01, or FSR_7.01, respectively. The invention alsocomprises other intervals genetically linked with those intervals.

Examples of markers useful for this purpose comprise the SNP markerslisted in Table 6, or any marker that maps within the chromosomeintervals described herein (including the termini of the intervals), orany marker linked to those markers. Such markers can be assayedsimultaneously or sequentially in a single sample or population ofsamples.

Accordingly, the markers and methods of the present invention can beutilized to guide MAS or breeding maize varieties with the desiredcomplement (set) of allelic forms of chromosome intervals associatedwith superior agronomic performance (resistance, along with any otheravailable markers for yield, disease resistance, etc.). Any of thedisclosed marker alleles can be introduced into a corn line viaintrogression, by traditional breeding (or introduced viatransformation, or both) to yield a corn plant with superior agronomicperformance. The number of alleles associated with resistance that canbe introduced or be present in a corn plant of the present inventionranges from one to the number of alleles disclosed herein, each integerof which is incorporated herein as if explicitly recited.

MAS using additional markers flanking either side of the DNA locusprovide further efficiency because an unlikely double recombinationevent would be needed to simultaneously break linkage between the locusand both markers. Moreover, using markers tightly flanking a locus, oneskilled in the art of MAS can reduce linkage drag by more accuratelyselecting individuals that have less of the potentially deleteriousdonor parent DNA. Any marker linked to or among the chromosome intervalsdescribed herein could be useful and within the scope of this invention.

Similarly, by identifying plants lacking the desired marker locus,susceptible or less resistant plants can be identified, and, e.g.,eliminated from subsequent crosses. Similarly, these marker loci can beintrogressed into any desired genomic background, germplasm, plant,line, variety, etc., as part of an overall MAS breeding program designedto enhance yield. The invention also provides chromosome QTL intervalsthat find equal use in MAS to select plants that demonstrate diseaseresistance or improved tolerance. Similarly, the QTL intervals can alsobe used to counter-select plants that are susceptible or have reducedresistance to disease.

The present invention also comprises methods of making a progeny cornplant and the progeny corn plants produced by these methods. The methodscomprise crossing a first parent corn plant with a second corn plant andgrowing the female corn plant under plant growth conditions to yieldcorn plant progeny. Methods of crossing and growing corn plants are wellwithin the ability of those of ordinary skill in the art. Such cornplant progeny can be assayed for alleles associated with resistance and,thereby, the desired progeny selected. Such progeny plants or seed canbe sold commercially for corn production, used for food, processed toobtain a desired constituent of the corn, or further utilized insubsequent rounds of breeding. At least one of the first or second cornplants is a corn plant of the present invention in that it comprises atleast one of the allelic forms of the markers of the present invention,such that the progeny are capable of inheriting the allele.

Often, a method of the present invention is applied to at least onerelated corn plant such as from progenitor or descendant lines in thesubject corn plants' pedigree such that inheritance of the desiredresistance allele can be traced. The number of generations separatingthe corn plants being subject to the methods of the present inventionwill generally be from 1 to 20, commonly 1 to 5, and typically 1, 2, or3 generations of separation, and quite often a direct descendant orparent of the corn plant will be subject to the method (i.e., onegeneration of separation).

Thus, with this invention, one skilled in the art can detect thepresence or absence of disease resistance genotypes in the genomes ofcorn plants as part of a marker assisted selection program. In oneembodiment, a breeder ascertains the genotype at one or more markers fora disease resistant parent, which contains a disease resistance allele,and the genotype at one or more markers for a susceptible parent, whichlacks the resistance allele. For example, the markers of the presentinvention can be used in MAS in crosses involving elite x exotic cornlines by subjecting the segregating progeny to MAS to maintain diseaseresistance alleles, or alleles associated with yield under diseaseconditions. A breeder can then reliably track the inheritance of theresistance alleles through subsequent populations derived from crossesbetween the two parents by genotyping offspring with the markers used onthe parents and comparing the genotypes at those markers with those ofthe parents. Depending on how tightly linked the marker alleles are withthe trait, progeny that share genotypes with the disease resistantparent can be reliably predicted to express the resistant phenotype;progeny that share genotypes with the disease susceptible parent can bereliably predicted to express the susceptible phenotype. Thus, thelaborious and inefficient process of manually phenotyping the progenyfor disease resistance is avoided.

By providing the positions in the maize genome of the intervals and thedisease resistance associated markers within, this invention also allowsone skilled in the art to identify other markers within the intervalsdisclosed herein or linked to the chromosome intervals disclosed herein.

Closely linked markers flanking the locus of interest that have allelesin linkage disequilibrium with a resistance allele at that locus may beeffectively used to select for progeny plants with enhanced resistanceto disease conditions. Thus, the markers described herein, such as thoselisted in Tables 1a or 1b, as well as other markers genetically orphysically mapped to the same chromosome interval, may be used to selectfor maize plants with enhanced resistance to disease conditions.Typically, a set of these markers will be used, (e.g., 2 or more, 3 ormore, 4 or more, 5 or more) in the flanking region above the gene and asimilar set in the flanking region below the gene. Optionally, asdescribed above, a marker within the actual gene and/or locus may alsobe used. The parents and their progeny are screened for these sets ofmarkers, and the markers that are polymorphic between the two parentsare used for selection. In an introgression program, this allows forselection of the gene or locus genotype at the more proximal polymorphicmarkers and selection for the recurrent parent genotype at the moredistal polymorphic markers.

The choice of markers actually used to practice this invention is notparticularly limited and can be any marker that maps within theFSR_2.01, FSR_5.01, FSR_6.01, or FSR_7.01 chromosome intervals describedherein, any marker closely linked (within 10 cM) to a marker in theFSR_2.01, FSR_5.01, FSR_6.01, or FSR_7.01 chromosome intervals, or anymarker selected from SEQ ID NO: 1-27 and 136-154, or the markers listedin Table 6. Furthermore, since there are many different types of markerdetection assays known in the art, it is not intended that the type ofmarker detection assay (e.g. RAPDs, RFLPs, SNPs, AFLPs, etc.) used topractice this invention be limited in any way.

II. Molecular Genetic Markers

“Marker,” “genetic marker,” “molecular marker,” “marker nucleic acid,”and “marker locus” refer to a nucleotide sequence or encoded productthereof (e.g., a protein) used as a point of reference when identifyinga linked locus. A marker can be derived from genomic nucleotide sequenceor from expressed nucleotide sequences (e.g., from a spliced RNA, acDNA, etc.), or from an encoded polypeptide, and can be represented byone or more particular variant sequences, or by a consensus sequence. Inanother sense, a marker is an isolated variant or consensus of such asequence. The term also refers to nucleic acid sequences complementaryto or flanking the marker sequences, such as nucleic acids used asprobes or primer pairs capable of amplifying the marker sequence. A“marker probe” is a nucleic acid sequence or molecule that can be usedto identify the presence of a marker locus, e.g., a nucleic acid probethat is complementary to a marker locus sequence. Alternatively, in someaspects, a marker probe refers to a probe of any type that is able todistinguish (i.e., genotype) the particular allele that is present at amarker locus. A “marker locus” is a locus that can be used to track thepresence of a second linked locus, e.g., a linked locus that encodes orcontributes to expression of a phenotypic trait. For example, a markerlocus can be used to monitor segregation of alleles at a locus, such asa QTL, that are genetically or physically linked to the marker locus.Thus, a “marker allele,” alternatively an “allele of a marker locus” isone of a plurality of polymorphic nucleotide sequences found at a markerlocus in a population that is polymorphic for the marker locus.

“Marker” also refers to nucleic acid sequences complementary to thegenomic sequences, such as nucleic acids used as probes. Markerscorresponding to genetic polymorphisms between members of a populationcan be detected by methods well-established in the art. These include,e.g., PCR-based sequence specific amplification methods, detection ofrestriction fragment length polymorphisms (RFLP), detection of isozymemarkers, detection of polynucleotide polymorphisms by allele specifichybridization (ASH), detection of amplified variable sequences of theplant genome, detection of self-sustained sequence replication,detection of simple sequence repeats (SSRs), detection of singlenucleotide polymorphisms (SNPs), or detection of amplified fragmentlength polymorphisms (AFLPs). Well established methods are also know forthe detection of expressed sequence tags (ESTs) and SSR markers derivedfrom EST sequences and randomly amplified polymorphic DNA (RAPD).

A favorable allele of a marker is the allele of the marker thatco-segregates with a desired phenotype (e.g., disease resistance). Asused herein, a QTL marker has a minimum of one favorable allele,although it is possible that the marker might have two or more favorablealleles found in the population. Any favorable allele of that marker canbe used advantageously for the identification and construction ofdisease resistant plant lines. Optionally, one, two, three or morefavorable allele(s) of different markers are identified in, orintrogressed into a plant, and can be selected for or against duringMAS. Desirably, plants or germplasm are identified that have at leastone such favorable allele that positively correlates with diseaseresistance or improved disease resistance. Alternatively, a markerallele that co-segregates with disease susceptibility also finds usewith the invention, since that allele can be used to identify andcounter select disease susceptible plants. Such an allele can be usedfor exclusionary purposes during breeding to identify alleles thatnegatively correlate with resistance, to eliminate susceptible plants orgermplasm from subsequent rounds of breeding.

The more tightly linked a marker is with a DNA locus influencing aphenotype, the more reliable the marker is in MAS, as the likelihood ofa recombination event unlinking the marker and the locus decreases.Markers containing the causal mutation for a trait, or that are withinthe coding sequence of a causative gene, are ideal as no recombinationis expected between them and the sequence of DNA responsible for thephenotype.

Genetic markers are distinguishable from each other (as well as from theplurality of alleles of anyone particular marker) on the basis ofpolynucleotide length and/or sequence. A large number of corn molecularmarkers are known in the art, and are published or available fromvarious sources, such as the MaizeGDB internet resource. In general, anydifferentially inherited polymorphic trait (including a nucleic acidpolymorphism) that segregates among progeny is a potential geneticmarker.

In some embodiments of the invention, one or more marker alleles areselected for in a single plant or a population of plants. In thesemethods, plants are selected that contain favorable alleles from morethan one resistance marker, or alternatively, favorable alleles frommore than one resistance marker are introgressed into a desiredgermplasm. One of skill recognizes that the identification of favorablemarker alleles is germplasm-specific. The determination of which markeralleles correlate with resistance (or susceptibility) is determined forthe particular germplasm under study. One of skill recognizes thatmethods for identifying the favorable alleles are known in the art.Identification and use of such favorable alleles is within the scope ofthis invention. Furthermore still, identification of favorable markeralleles in plant populations other than the populations used ordescribed herein is within the scope of this invention.

Marker Detection

In some aspects, methods of the invention utilize an amplification stepto detect/genotype a marker locus, but amplification is not always arequirement for marker detection (e.g. Southern blotting and RFLPdetection). Separate detection probes can also be omitted inamplification/detection methods, e.g., by performing a real timeamplification reaction that detects product formation by modification ofthe relevant amplification primer upon incorporation into a product,incorporation of labeled nucleotides into an amplicon, or by monitoringchanges in molecular rotation properties of amplicons as compared tounamplified precursors (e.g., by fluorescence polarization).

“Amplifying,” in the context of nucleic acid amplification, is anyprocess whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. In some embodiments, anamplification based marker technology is used wherein a primer oramplification primer pair is admixed with genomic nucleic acid isolatedfrom the first plant or germplasm, and wherein the primer or primer pairis complementary or partially complementary to at least a portion of themarker locus, and is capable of initiating DNA polymerization by a DNApolymerase using the plant genomic nucleic acid as a template. Theprimer or primer pair is extended in a DNA polymerization reactionhaving a DNA polymerase and a template genomic nucleic acid to generateat least one amplicon. In other embodiments, plant RNA is the templatefor the amplification reaction. In some embodiments, the QTL marker is aSNP type marker, and the detected allele is a SNP allele, and the methodof detection is allele specific hybridization (ASH).

In general, the majority of genetic markers rely on one or more propertyof nucleic acids for their detection. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods. An “amplicon” is an amplified nucleicacid, e.g., a nucleic acid that is produced by amplifying a templatenucleic acid by any available amplification method (e.g., PCR, LCR,transcription, or the like). A “genomic nucleic acid” is a nucleic acidthat corresponds in sequence to a heritable nucleic acid in a cell.Common examples include nuclear genomic DNA and amplicons thereof. Agenomic nucleic acid is, in some cases, different from a spliced RNA, ora corresponding cDNA, in that the spliced RNA or cDNA is processed,e.g., by the splicing machinery, to remove introns. Genomic nucleicacids optionally comprise non-transcribed (e.g., chromosome structuralsequences, promoter regions, enhancer regions, etc.) and/ornon-translated sequences (e.g., introns), whereas spliced RNA/cDNAtypically do not have non-transcribed sequences or introns. A “templatenucleic acid” is a nucleic acid that serves as a template in anamplification reaction (e.g., a polymerase based amplification reactionsuch as PCR, a ligase mediated amplification reaction such as LCR, atranscription reaction, or the like). A template nucleic acid can begenomic in origin, or alternatively, can be derived from expressedsequences, e.g., a cDNA or an EST. Details regarding the use of theseand other amplification methods can be found in any of a variety ofstandard texts. Many available biology texts also have extendeddiscussions regarding PCR and related amplification methods and one ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase.

PCR detection and quantification using dual-labeled fluorogenicoligonucleotide probes, commonly referred to as “TaqMan™” probes, canalso be performed according to the present invention. These probes arecomposed of short (e.g., 20-25 base) oligodeoxynucleotides that arelabeled with two different fluorescent dyes. On the 5′ terminus of eachprobe is a reporter dye, and on the 3′ terminus of each probe aquenching dye is found. The oligonucleotide probe sequence iscomplementary to an internal target sequence present in a PCR amplicon.When the probe is intact, energy transfer occurs between the twofluorophores and emission from the reporter is quenched by the quencherby FRET. During the extension phase of PCR, the probe is cleaved by 5′nuclease activity of the polymerase used in the reaction, therebyreleasing the reporter from the oligonucleotide-quencher and producingan increase in reporter emission intensity. TaqMan™ probes areoligonucleotides that have a label and a quencher, where the label isreleased during amplification by the exonuclease action of thepolymerase used in amplification, providing a real time measure ofamplification during synthesis. A variety of TaqMan™ reagents arecommercially available, e.g., from Applied Biosystems as well as from avariety of specialty vendors such as Biosearch Technologies.

In one embodiment, the presence or absence of a molecular marker isdetermined simply through nucleotide sequencing of the polymorphicmarker region. This method is readily adapted to high throughputanalysis as are the other methods noted above, e.g., using availablehigh throughput sequencing methods such as sequencing by hybridization.

In alternative embodiments, in silico methods can be used to detect themarker loci of interest. For example, the sequence of a nucleic acidcomprising the marker locus of interest can be stored in a computer. Thedesired marker locus sequence or its homolog can be identified using anappropriate nucleic acid search algorithm as provided by, for example,in such readily available programs as BLAST, or even simple wordprocessors.

While the exemplary markers provided in the figures and tables hereinare either SNP markers, any of the aforementioned marker types can beemployed in the context of the invention to identify chromosomeintervals encompassing genetic element that contribute to superioragronomic performance (e.g., disease resistance or improved diseasetolerance).

Probes and Primers

In general, synthetic methods for making oligonucleotides, includingprobes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids),etc., are known. For example, oligonucleotides can be synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed. Oligonucleotides, including modified oligonucleotides, canalso be ordered from a variety of commercial sources.

Nucleic acid probes to the marker loci can be cloned and/or synthesized.Any suitable label can be used with a probe of the invention. Detectablelabels suitable for use with nucleic acid probes include, for example,any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels include biotin for staining with labeledstreptavidin conjugate, magnetic beads, fluorescent dyes, radio labels,enzymes, and colorimetric labels. Other labels include ligands whichbind to antibodies labeled with fluorophores, chemiluminescent agents,and enzymes. A probe can also constitute radio labeled PCR primers thatare used to generate a radio labeled amplicon. It is not intended thatthe nucleic acid probes of the invention be limited to any particularsize.

In some preferred embodiments, the molecular markers of the inventionare detected using a suitable PCR-based detection method, where the sizeor sequence of the PCR amplicon is indicative of the absence or presenceof the marker (e.g., a particular marker allele). In these types ofmethods, PCR primers are hybridized to the conserved regions flankingthe polymorphic marker region. As used in the art, PCR primers used toamplify a molecular marker are sometimes termed “PCR markers” or simply“markers.” It will be appreciated that, although many specific examplesof primers are provided herein, suitable primers to be used with theinvention can be designed using any suitable method. It is not intendedthat the invention be limited to any particular primer or primer pair.In some embodiments, the primers of the invention are radiolabelled, orlabeled by any suitable means (e.g., using a non-radioactive fluorescenttag), to allow for rapid visualization of the different size ampliconsfollowing an amplification reaction without any additional labeling stepor visualization step. In some embodiments, the primers are not labeled,and the amplicons are visualized following their size resolution, e.g.,following agarose gel electrophoresis. In some embodiments, ethidiumbromide staining of the PCR amplicons following size resolution allowsvisualization of the different size amplicons. It is not intended thatthe primers of the invention be limited to generating an amplicon of anyparticular size. For example, the primers used to amplify the markerloci and alleles herein are not limited to amplifying the entire regionof the relevant locus. The primers can generate an amplicon of anysuitable length that is longer or shorter than those disclosed herein.In some embodiments, marker amplification produces an amplicon at least20 nucleotides in length, or alternatively, at least 50 nucleotides inlength, or alternatively, at least 100 nucleotides in length, oralternatively, at least 200 nucleotides in length. Marker alleles inaddition to those recited herein also find use with the presentinvention.

III. Linkage Analysis and QTL Linkage Analysis

“Linkage”, or “genetic linkage,” is used to describe the degree withwhich one marker locus is “associated with” another marker locus or someother locus (for example, a resistance locus). For example, if locus Ahas genes “A” or “a” and locus B has genes “B” or “b” and a crossbetween parent 1 with AABB and parent 2 with aabb will produce fourpossible gametes where the genes are segregated into AB, Ab, aB and ab.The null expectation is that there will be independent equal segregationinto each of the four possible genotypes, i.e. with no linkage ¼ of thegametes will of each genotype. Segregation of gametes into a genotypesdiffering from ¼ is attributed to linkage. As used herein, linkage canbe between two markers, or alternatively between a marker and aphenotype. A marker locus can be associated with (linked to) a trait,e.g., a marker locus can be associated with resistance or improvedtolerance to a plant pathogen when the marker locus is in linkagedisequilibrium (LD) with the resistance trait. The degree of linkage ofa molecular marker to a phenotypic trait (e.g., a QTL) is measured,e.g., as a statistical probability of co-segregation of that molecularmarker with the phenotype.

As used herein, the linkage relationship between a molecular marker anda phenotype is given is the statistical likelihood that the particularcombination of a phenotype and the presence or absence of a particularmarker allele is random. Thus, the lower the probability score, thegreater the likelihood that a phenotype and a particular marker willcosegregate. In some embodiments, a probability score of 0.05 (p=0.05,or a 5% probability) of random assortment is considered a significantindication of co-segregation. However, the present invention is notlimited to this particular standard, and an acceptable probability canbe any probability of less than 50% (p<0.5). For example, a significantprobability can be less than 0.25, less than 0.20, less than 0.15, orless than 0.1. The phrase “closely linked,” in the present application,means that recombination between two linked loci occurs with a frequencyof equal to or less than about 10% (i.e., are separated on a genetic mapby not more than 10 cM). In one aspect, any marker of the invention islinked (genetically and physically) to any other marker that is at orless than 50 cM distant. In another aspect, any marker of the inventionis closely linked (genetically and physically) to any other marker thatis in close proximity, e.g., at or less than 10 cM distant. Two closelylinked markers on the same chromosome can be positioned 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25cM or less from each other.

Classical linkage analysis can be thought of as a statisticaldescription of the relative frequencies of co segregation of differenttraits. Linkage analysis is the well characterized descriptive frameworkof how traits are grouped together based upon the frequency with whichthey segregate together. That is, if two non-allelic traits areinherited together with a greater than random frequency, they are saidto be “linked.” The frequency with which the traits are inheritedtogether is the primary measure of how tightly the traits are linked,i.e., traits which are inherited together with a higher frequency aremore closely linked than traits which are inherited together with lower(but still above random) frequency. The further apart on a chromosomethe genes reside, the less likely they are to segregate together,because homologous chromosomes recombine during meiosis. Thus, thefurther apart on a chromosome the genes reside, the more likely it isthat there will be a crossing over event during meiosis that will resultin the marker and the DNA sequence responsible for the trait the markeris designed to track segregating separately into progeny. A commonmeasure of linkage is the frequency with which traits cosegregate. Thiscan be expressed as a percentage of cosegregation (recombinationfrequency) or, also commonly, in centiMorgans (cM).

Linkage analysis is used to determine which polymorphic marker alleledemonstrates a statistical likelihood of co-segregation with theresistance phenotype (thus, a “resistance marker allele”). Followingidentification of a marker allele for co-segregation with the resistancephenotype, it is possible to use this marker for rapid, accuratescreening of plant lines for the resistance allele without the need togrow the plants through their life cycle and await phenotypicevaluations, and furthermore, permits genetic selection for theparticular resistance allele even when the molecular identity of theactual resistance QTL is unknown. Tissue samples can be taken, forexample, from the endosperm, embryo, or mature/developing plant andscreened with the appropriate molecular marker to rapidly determinedetermined which progeny contain the desired genetics Linked markersalso remove the impact of environmental factors that can often influencephenotypic expression.

Because chromosomal distance is approximately proportional to thefrequency of crossing over events between traits, there is anapproximate physical distance that correlates with recombinationfrequency. Marker loci are themselves traits and can be assessedaccording to standard linkage analysis by tracking the marker lociduring segregation. Thus, in the context of the present invention, onecM is equal to a 1% chance that a marker locus will be separated fromanother locus (which can be any other trait, e.g., another marker locus,or another trait locus that encodes a QTL), due to crossing over in asingle generation.

When referring to the relationship between two genetic elements, such asa genetic element contributing to resistance and a proximal marker,“coupling” phase linkage indicates the state where the “favorable”allele at the resistance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest (e.g., aQTL for resistance) is physically linked with an “unfavorable” allele atthe proximal marker locus, and the two “favorable” alleles are notinherited together (i.e., the two loci are “out of phase” with eachother).

Quantitative Trait Loci

An allele of a QTL can comprise multiple genes or other genetic factorseven within a contiguous genomic region or linkage group, such as ahaplotype. As used herein, an allele of a disease resistance locus canencompass more than one gene or nucleotide sequence where eachindividual gene or nucleotide sequence is also capable of exhibitingallelic variation and where each gene or nucleotide sequence is alsocapable of eliciting a phenotypic effect on the quantitative trait inquestion. In an aspect of the present invention the allele of a QTLcomprises one or more genes or nucleic acid sequences that are alsocapable of exhibiting allelic variation. The use of the term “an alleleof a QTL” is thus not intended to exclude a QTL that comprises more thanone gene or other genetic factor. Specifically, an “allele of a QTL” inthe present in the invention can denote a haplotype within a haplotypewindow wherein a phenotype can be disease resistance. A haplotype windowis a contiguous genomic region that can be defined, and tracked, with aset of one or more polymorphic markers wherein the polymorphismsindicate identity by descent. A haplotype within that window can bedefined by the unique fingerprint of alleles at each marker. When allthe alleles present at a given locus on a chromosome are the same, thatplant is homozygous at that locus. If the alleles present at a givenlocus on a chromosome differ, that plant is heterozygous at that locus.Plants of the present invention may be homozygous or heterozygous at anyparticular disease locus or for a particular polymorphic marker.

The principles of QTL analysis and statistical methods for calculatinglinkage between markers and useful QTL, or between any loci in a genomeare well known in the art. Exemplary methods include penalizedregression analysis, ridge regression, single point marker analysis,complex pedigree analysis, Bayesian MCMC, identity-by-descent analysis,interval mapping, composite interval mapping, and Haseman-Elstonregression. QTL analyses are often performed with the help of a computerand specialized software available from a variety of public andcommercial sources known to those of skill in the art.

IV. Genetic Mapping

A “genetic map” is the relationship of genetic linkage among loci on oneor more chromosomes (or linkage groups) within a given species,generally depicted in a diagrammatic or tabular form. “Genetic mapping”is the process of defining the linkage relationships of loci through theuse of genetic markers, populations segregating for the markers, andstandard genetic principles of recombination frequency. A “genetic maplocation” is a location on a genetic map relative to surrounding geneticmarkers on the same linkage group where a specified marker can be foundwithin a given species. In contrast, a physical map of the genome refersto absolute distances (for example, measured in base pairs or isolatedand overlapping contiguous genetic fragments, e.g., contigs). A physicalmap of the genome does not take into account the genetic behavior (e.g.,recombination frequencies) between different points on the physical map.A “genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis. A genetic recombination frequency can be expressed incentimorgans (cM). In some cases, two different markers can have thesame genetic map coordinates. In that case, the two markers are in suchclose proximity to each other that recombination occurs between themwith such low frequency that it is undetected.

Genetic maps are graphical representations of genomes (or a portion of agenome such as a single chromosome) where the distances between markersare measured by the recombination frequencies between them. Plantbreeders use genetic maps of molecular markers to increase breedingefficiency through Marker assisted selection (MAS), a process whereselection for a trait of interest is not based on the trait itself butrather on the genotype of a marker linked to the trait. A molecularmarker that demonstrates reliable linkage with a phenotypic traitprovides a useful tool for indirectly selecting the trait in a plantpopulation, especially when accurate phenotyping is difficult, slow, orexpensive.

In general, the closer two markers or genomic loci are on the geneticmap, the closer they lie to one another on the physical map. A lack ofprecise proportionality between cM distances and physical distances canexist due to the fact that the likelihood of genetic recombination isnot uniform throughout the genome; some chromosome regions arecross-over “hot spots,” while other regions demonstrate only rarerecombination events, if any.

Genetic mapping variability can also be observed between differentpopulations of the same crop species. In spite of this variability inthe genetic map that may occur between populations, genetic map andmarker information derived from one population generally remains usefulacross multiple populations in identification of plants with desiredtraits, counter-selection of plants with undesirable traits and inguiding MAS.

As one of skill in the art will recognize, recombination frequencies(and as a result, genetic map positions) in any particular populationare not static. The genetic distances separating two markers (or amarker and a QTL) can vary depending on how the map positions aredetermined. For example, variables such as the parental mappingpopulations used, the software used in the marker mapping or QTLmapping, and the parameters input by the user of the mapping softwarecan contribute to the QTL marker genetic map relationships. However, itis not intended that the invention be limited to any particular mappingpopulations, use of any particular software, or any particular set ofsoftware parameters to determine linkage of a particular marker orchromosome interval with the disease resistance phenotype. It is wellwithin the ability of one of ordinary skill in the art to extrapolatethe novel features described herein to any gene pool or population ofinterest, and using any particular software and software parameters.Indeed, observations regarding genetic markers and chromosome intervalsin populations in addition to those described herein are readily madeusing the teaching of the present disclosure.

Association Mapping

Association or LD mapping techniques aim to identify genotype-phenotypeassociations that are significant. It is effective for fine mapping inoutcrossing species where frequent recombination among heterozygotes canresult in rapid LD decay. LD is non-random association of alleles in acollection of individuals, reflecting the recombinational history ofthat region. Thus, LD decay averages can help determine the number ofmarkers necessary for a genome-wide association study to generate agenetic map with a desired level of resolution.

Large populations are better for detecting recombination, while olderpopulations are generally associated with higher levels of polymorphism,both of which contribute to accelerated LD decay. However, smallereffective population sizes tend to show slower LD decay, which canresult in more extensive haplotype conservation. Understanding of therelationships between polymorphism and recombination is useful indeveloping strategies for efficiently extracting information from theseresources. Association analyses compare the plants' phenotypic scorewith the genotypes at the various loci. Subsequently, any suitable maizegenetic map (for example, a composite map) can be used to help observedistribution of the identified QTL markers and/or QTL marker clusteringusing previously determined map locations of the markers.

V. Marker Assisted Selection, Plant Breeding, and Genomic IntrogressionMarker Assisted Selection

“Introgression” refers to the transmission of a desired allele of agenetic locus from one genetic background to another by. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g., aselected allele of a marker, a QTL, a transgene, or the like. In anycase, offspring comprising the desired allele can be repeatedlybackcrossed to a line having a desired genetic background and selectedfor the desired allele, to result in the allele becoming fixed in aselected genetic background.

A primary motivation for development of molecular markers in cropspecies is the potential for increased efficiency in plant breedingthrough marker assisted selection (MAS). Genetic markers are used toidentify plants that contain a desired genotype at one or more loci, andthat are expected to transfer the desired genotype, along with a desiredphenotype to their progeny. Genetic markers can be used to identifyplants containing a desired genotype at one locus, or at severalunlinked or linked loci (e.g., a haplotype), and that would be expectedto transfer the desired genotype, along with a desired phenotype totheir progeny. The present invention provides the means to identifyplants that are resistant, exhibit improved resistance or aresusceptible to FSR infection by identifying plants having a specifiedallele that is linked to FSR_2.01, FSR_5.01, FSR_6.01, or FSR_7.01.

In general, MAS uses polymorphic markers that have been identified ashaving a significant likelihood of co-segregation with a resistancetrait. Such markers are presumed to map near a gene or genes that givethe plant its resistance phenotype, and are considered indicators forthe desired trait, and are termed QTL markers. Plants are tested for thepresence or absence of a desired allele in the QTL marker.

Identification of plants or germplasm that include a marker locus ormarker loci linked to a resistance trait or traits provides a basis forperforming marker assisted selection. Plants that comprise favorablemarkers or favorable alleles are selected for, while plants thatcomprise markers or alleles that are negatively correlated withresistance can be selected against. Desired markers and/or alleles canbe introgressed into plants having a desired (e.g., elite or exotic)genetic background to produce an introgressed resistant plant orgermplasm. In some aspects, it is contemplated that a plurality ofresistance markers are sequentially or simultaneous selected and/orintrogressed. The combinations of resistance markers that are selectedfor in a single plant is not limited, and can include any combination ofmarkers disclosed herein or any marker linked to the markers disclosedherein, or any markers located within the QTL intervals defined herein.

In some embodiments, the allele that is detected is a favorable allelethat positively correlates with disease resistance or improved diseasetolerance. In the case where more than one marker is selected, an alleleis selected for each of the markers; thus, two or more alleles areselected. In some embodiments, it can be the case that a marker locuswill have more than one advantageous allele, and in that case, eitherallele can be selected. It will be appreciated that the ability toidentify QTL marker loci alleles that correlate with resistance,improved tolerance, or susceptibility of a corn plant to diseaseconditions provides a method for selecting plants that have favorablemarker loci as well. That is, any plant that is identified as comprisinga desired marker locus (e.g., a marker allele that positively correlateswith resistance) can be selected for, while plants that lack the locus,or that have a locus that negatively correlates with resistance, can beselected against.

In some embodiments, a disease resistant first corn plant or germplasm(the donor) can be crossed with a second corn plant or germplasm (therecipient, e.g., an elite or exotic corn, depending on characteristicsthat are desired in the progeny) to create an introgressed corn plant orgermplasm as part of a breeding program designed to improve diseaseresistance of the recipient corn plant or germplasm. In some aspects,the recipient plant can also contain one or more disease resistant loci,which can be qualitative or quantitative trait loci. In another aspect,the recipient plant can contain a transgene.

In some embodiments, the recipient corn plant or germplasm willtypically display reduced resistance to disease conditions as comparedto the first corn plant or germplasm, while the introgressed corn plantor germplasm will display an increased resistance to disease conditionsas compared to the second plant or germplasm. An introgressed corn plantor germplasm produced by these methods are also a feature of thisinvention.

MAS is a powerful shortcut to selecting for desired phenotypes and forintrogressing desired traits into cultivars (e.g., introgressing desiredtraits into elite lines). MAS is easily adapted to high throughputmolecular analysis methods that can quickly screen large numbers ofplant or germplasm genetic material for the markers of interest and ismuch more cost effective than raising and observing plants for visibletraits.

When a population is segregating for multiple loci affecting one ormultiple traits, e.g., multiple loci involved in resistance, or multipleloci each involved in resistance or tolerance to different diseases, theefficiency of MAS compared to phenotypic screening becomes even greater,because all of the loci can be evaluated in the lab together from asingle sample of DNA.

Marker Assisted Backcrossing

One application of MAS is to use the resistance or improved tolerancemarkers to increase the efficiency of an introgression effort aimed atintroducing a resistance QTL into a desired (typically high yielding)background. If the nucleic acids from a plant are positive for a desiredgenetic marker allele, the plant can be self-fertilized to create a truebreeding line with the same genotype, or it can be crossed with a plantwith the same marker or with other characteristics to create a sexuallycrossed hybrid generation.

Another use of MAS in plant breeding is to assist the recovery of therecurrent parent genotype by backcross breeding. Backcross breeding isthe process of crossing a progeny back to one of its parents or parentlines. Backcrossing is usually done for the purpose of introgressing oneor a few loci from a donor parent (e.g., a parent comprising desirableresistance marker loci) into an otherwise desirable genetic backgroundfrom the recurrent parent (e.g., an otherwise high yielding line). Themore cycles of back crossing that are done, the greater the geneticcontribution of the recurrent parent to the resulting introgressedvariety. This is often necessary, because resistant plants may beotherwise undesirable, e.g., due to low yield, low fecundity, or thelike. In contrast, strains which are the result of intensive breedingprograms may have excellent yield, fecundity or the like, merely beingdeficient in one desired trait such as resistance to FSR infection.

Moreover, in another aspect, while maintaining the introduced markersassociated with resistance, the genetic contribution of the plantproviding disease resistance can be reduced by back-crossing or othersuitable approaches. In one aspect, the nuclear genetic material derivedfrom the donor material in the plant can be less than or about 50%, lessthan or about 25%, less than or about 13%, less than or about 5%, 3%, 2%or 1%, but that the recipient remains resistant to disease.

Genetic diversity is important for long term genetic gain in anybreeding program. With limited diversity, genetic gain will eventuallyplateau when all of the favorable alleles have been fixed within theelite population. One objective is to incorporate diversity into anelite pool without losing the genetic gain that has already been madeand with the minimum possible investment. MAS provide an indication ofwhich genomic regions and which favorable alleles from the originalancestors have been selected for and conserved over time, facilitatingefforts to incorporate favorable variation from exotic germplasm sources(parents that are unrelated to the elite gene pool) in the hopes offinding favorable alleles that do not currently exist in the elite genepool.

Genomic Selection

Genomic selection (GS), also known as genome wide selection (GWS), is aform of MAS that estimates all locus, haplotype, and/or marker effectsacross the entire genome to calculate genomic estimated breeding values(GEBVs). See Nakaya and Isobe, Annals of Botany 110: 1303-1316 (2012);Van Vleck, et al., Journal of Animal Science 70: 363-371 (1992); andHeffner, et al., Crop Science 49: 1-12 (2009). GS utilizes a trainingphase and a breeding phase. In the training phase, genotypes andphenotypes are analyzed in a subset of a population to generate a GSprediction model that incorporates significant relationships betweenphenotypes and genotypes. A GS training population must berepresentative of selection candidates in the breeding program to whichGS will be applied. In the breeding phase, genotype data are obtained ina breeding population, then favorable individuals are selected based onGEBVs obtained using the GS prediction model generated during thetraining phase without the need for phenotypic data.

Larger training populations typically increase the accuracy of GEBVpredictions. Increasing the training population to breeding populationratio is helpful for obtaining accurate GEBVs when working withpopulations having high genetic diversity, small breeding populations,low heritability of traits, or large numbers of QTLs. The number ofmarkers required for GS modeling is determined based on the rate of LDdecay across the genome, which must be calculated for each specificpopulation to which GS will be applied. In general, more markers will benecessary with faster raters of LD decay. Ideally, GS comprises at leastone marker in LD with each QTL, but in practical terms one of ordinaryskill in the art would recognize that this is not necessary.

With genotyping data, favorable individuals from a population can beselected based only on GEBVs. GEBVs are the sum of the estimate ofgenetic deviation and the weighted sum of estimates of breed effects,which are predicted using phenotypic data. Without being limiting,commonly used statistical models for prediction of GEBVs include bestlinear unbiased prediction (Henderson, Biometrics 31: 423 (1975)) and aBayesian framework (Gianola and Fernando, Journal of Animal Science 63:217-244 (1986)).

The compositions and methods of the present disclosure can be utilizedfor GS or breeding corn varieties with a desired complement (set) ofallelic forms of chromosome intervals associated with superior agronomicperformance (e.g., FSR resistance). In an aspect, a corn plant or seedprovided herein can be selected using genomic selection. In anotheraspect, SEQ ID NOs: 1-27 and 136-154 can be used in a method comprisinggenomic selection. In another aspect, a genomic selection methodprovided herein comprises phenotyping a population of corn plants forFSR resistance using the FSR infection rating scale provided in Table 1.In another aspect, a genomic selection method provided herein comprisesgenotyping a population of corn plants or seeds with at least one ofmarker loci SEQ ID NOs: 1-27 and 136-154.

VI. Transgenic Plants Transformation Constructs

Vectors used for plant transformation may include, for example,plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterialartificial chromosomes) or any other suitable cloning system, as well asfragments of DNA therefrom. Thus when the term “vector” or “expressionvector” is used, all of the foregoing types of vectors, as well asnucleic acid sequences isolated therefrom, are included. It iscontemplated that utilization of cloning systems with large insertcapacities will allow introduction of large DNA sequences comprisingmore than one selected gene. In accordance with the present disclosure,this could be used to introduce genes corresponding to, e.g., an entirebiosynthetic pathway, into a plant.

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will generally comprise the cDNA, gene, or genes which onedesires to introduce into and have expressed in the host cells. TheseDNA segments can further include structures such as promoters,enhancers, polylinkers, or regulatory genes as desired. The DNA segmentor gene chosen for cellular introduction will often encode a proteinwhich will be expressed in the resultant recombinant cells resulting ina screenable or selectable trait and/or which will impart an improvedphenotype to the resulting transgenic plant.

Regulatory elements such as promoters, leaders, enhancers, introns, andtranscription termination regions (or 3′ UTRs) can play an integral partin the overall expression of genes in living cells. The term “regulatoryelement,” as used herein, refers to a DNA molecule havinggene-regulatory activity. The term “gene-regulatory activity,” as usedherein, refers to the ability to affect the expression of an operablylinked transcribable DNA molecule, for instance by affecting thetranscription and/or translation of the operably linked transcribableDNA molecule. Regulatory elements, such as promoters, leaders,enhancers, and introns that function in plants are therefore useful formodifying plant phenotypes through genetic engineering.

As used herein, the term “intron” refers to a DNA molecule that may beisolated or identified from the genomic copy of a gene and may bedefined generally as a region spliced out during messenger RNA (mRNA)processing prior to translation. Alternately, an intron may be asynthetically produced or manipulated DNA element. An intron may containenhancer elements that effect the transcription of operably linkedgenes. An intron may be used as a regulatory element for modulatingexpression of an operably linked transcribable DNA molecule. A constructmay comprise an intron, and the intron may or may not be heterologouswith respect to the transcribable DNA molecule. Examples of introns inthe art include the rice actin intron and the corn HSP70 intron. Inplants, the inclusion of some introns in constructs leads to increasedmRNA and protein accumulation relative to constructs lacking the intron.This effect has been termed “intron mediated enhancement” (IME) of geneexpression. Introns known to stimulate expression in plants have beenidentified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), in ricegenes (e.g., tpi) and in dicotyledonous plant genes like those frompetunia (e.g., rbcS), potato (e.g., st-isI) and from Arabidopsisthaliana (e.g., ubq3 and pat1). It has been shown that deletions ormutations within the splice sites of an intron reduce gene expression,indicating that splicing might be needed for IME. However, that splicingper se is not required, as IME in dicotyledonous plants has been shownby point mutations within the splice sites of the pat1 gene from A.thaliana. Multiple uses of the same intron in one plant have been shownto exhibit disadvantages. In those cases, it is necessary to have acollection of basic control elements for the construction of appropriaterecombinant DNA elements.

As used herein, the term “enhancer” or “enhancer element” refers to acis-acting regulatory element, a.k.a. cis-element, which confers anaspect of the overall expression pattern, but is usually insufficientalone to drive transcription, of an operably linked DNA sequence. Unlikepromoters, enhancer elements do not usually include a transcriptionstart site (TSS) or TATA box or equivalent DNA sequence. A promoter orpromoter fragment may naturally comprise one or more enhancer elementsthat affect the transcription of an operably linked DNA sequence. Anenhancer element may also be fused to a promoter to produce a chimericpromoter cis-element, which confers an aspect of the overall modulationof gene expression.

Regulatory elements may be characterized by their gene expressionpattern, e.g., positive and/or negative effects, such as constitutiveexpression or temporal, spatial, developmental, tissue, environmental,physiological, pathological, cell cycle, and/or chemically responsiveexpression, and any combination thereof, as well as by quantitative orqualitative indications. As used herein, a “gene expression pattern” isany pattern of transcription of an operably linked DNA molecule into atranscribed RNA molecule. The transcribed RNA molecule may be translatedto produce a protein molecule or may provide an antisense or otherregulatory RNA molecule, such as a double-stranded RNA (dsRNA), atransfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), and thelike.

As used herein, the term “protein expression” is any pattern oftranslation of a transcribed RNA molecule into a protein molecule.Protein expression may be characterized by its temporal, spatial,developmental, or morphological qualities, as well as by quantitative orqualitative indications.

A promoter is useful as a regulatory element for modulating theexpression of an operably linked transcribable DNA molecule. As usedherein, the term “promoter” refers generally to a DNA molecule that isinvolved in recognition and binding of RNA polymerase II and otherproteins, such as trans-acting transcription factors, to initiatetranscription. A promoter may originate from the 5′ untranslated region(5′ UTR) of a gene. Alternately, promoters may be synthetically producedor manipulated DNA molecules. Promoters may also be chimeric. As usedherein, the term “chimeric” refers to a single DNA molecule produced byfusing a first DNA molecule to a second DNA molecule, where neither thefirst nor the second DNA molecule would normally be contained in thatconfiguration, i.e., fused to the other. The chimeric DNA molecule isthus a new DNA molecule not otherwise normally contained in nature. Asused herein, the term “chimeric promoter” refers to a promoter producedthrough such manipulation of DNA molecules. A chimeric promoter maycombine two or more DNA fragments, for example, the fusion of a promoterto an enhancer element. Thus, the design, construction, and use ofchimeric promoters according to the methods disclosed herein formodulating the expression of operably linked transcribable DNA moleculesare encompassed by the disclosure.

In specific embodiments, chimeric DNA molecules and any variants orderivatives thereof as described herein, are further defined ascomprising promoter activity, i.e., are capable of acting as a promoterin a host cell, such as in a transgenic plant. In still further specificembodiments, a fragment may be defined as exhibiting promoter activitypossessed by the starting promoter molecule from which it is derived, ora fragment may comprise a “minimal promoter” which provides a basallevel of transcription and is comprised of a TATA box or equivalent DNAsequence for recognition and binding of the RNA polymerase II complexfor initiation of transcription.

Exemplary promoters for expression of a nucleic acid sequence includeplant promoters such as the CaMV 35S promoter, or others such as CaMV19S, nos, Adh, sucrose synthase, α-tubulin, actin, cab, PEPCase or thosepromoters associated with the R gene complex. Tissue-specific promoterssuch as leaf specific promoters, or tissue selective promoters (e.g.,promoters that direct greater expression in leaf primordia than in othertissues), and tissue-specific enhancers are also contemplated to beuseful, as are inducible promoters such as ABA- and turgor-induciblepromoters. Any suitable promoters known in the art may be used toexpress defensin or defensin-like coding sequences in a plant. In anembodiment, the CaMV35S promoter may be used to express defensin ordefensin-like coding sequences in a plant. In yet another embodiment, adisease or pathogen inducible promoter can be used to express defensinor defensin like proteins. Examples of disease or pathogen induciblepromoters can be found in Kooshki et al. Plant Science 165 (2003)213-219, Koschmann et al. Plant Physiology 160 (2012) 178-191, Rushtonet al. The Plant Cell, 14 (2002) 749-762, and Kirsch et al. The PlantJournal (2001) 26 217-227.

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. As used herein, the term “leader” refers to aDNA molecule from the untranslated 5′ region (5′ UTR) of a gene anddefined generally as a DNA segment between the transcription start site(TSS) and the protein coding sequence start site. Alternately, leadersmay be synthetically produced or manipulated DNA elements. A leader canbe used as a 5′ regulatory element for modulating expression of anoperably linked transcribable DNA molecule. Leader molecules may be usedwith a heterologous promoter or with their native promoter. One may thuswish to employ a particular leader sequence with a transformationconstruct of the present disclosure. In an embodiment, leader sequencesare contemplated to include those which comprise sequences predicted todirect optimum expression of the attached gene, i.e., to include aconsensus leader sequence which may increase or maintain mRNA stabilityand prevent inappropriate initiation of translation. The choice of suchsequences will be known to those of skill in the art in light of thepresent disclosure. In some embodiments, sequences that are derived fromgenes that are highly expressed in plants may be used for expression ofdefensin or defensin-like coding sequences.

Transformation constructs prepared in accordance with the presentdisclosure may further include a 3′ end DNA sequence that acts as asignal to terminate transcription and allow for the polyadenylation ofthe mRNA produced by coding sequences operably linked to a promoter. Asused herein, the term “3′ transcription termination molecule,” “3′untranslated region” or “3′ UTR” herein refers to a DNA molecule that isused during transcription to the untranslated region of the 3′ portionof an mRNA molecule. The 3′ untranslated region of an mRNA molecule maybe generated by specific cleavage and 3′ polyadenylation, also known asa polyA tail. A 3′ UTR may be operably linked to and located downstreamof a transcribable DNA molecule and may include a polyadenylation signaland other regulatory signals capable of affecting transcription, mRNAprocessing, or gene expression. PolyA tails are thought to function inmRNA stability and in initiation of translation. Examples of 3′transcription termination molecules in the art are the nopaline synthase3′ region; wheat hsp17 3′ region, pea rubisco small subunit 3′ region,cotton E6 3′ region, and the coixin 3′ UTR.

3′ UTRs typically find beneficial use for the recombinant expression ofspecific DNA molecules. A weak 3′ UTR has the potential to generateread-through, which may affect the expression of the DNA moleculelocated in the neighboring expression cassettes. Appropriate control oftranscription termination can prevent read-through into DNA sequences(e.g., other expression cassettes) localized downstream and can furtherallow efficient recycling of RNA polymerase to improve gene expression.Efficient termination of transcription (release of RNA Polymerase IIfrom the DNA) is prerequisite for re-initiation of transcription andthereby directly affects the overall transcript level. Subsequent totranscription termination, the mature mRNA is released from the site ofsynthesis and template transported to the cytoplasm. Eukaryotic mRNAsare accumulated as poly(A) forms in vivo, making it difficult to detecttranscriptional termination sites by conventional methods. However,prediction of functional and efficient 3′ UTRs by bioinformatics methodsis difficult in that there are no conserved DNA sequences that wouldallow easy prediction of an effective 3′ UTR. In one embodiment, thenative terminator of a defensin or defensin-like coding sequence may beused. Alternatively, a heterologous 3′ end may enhance the expression ofsense or antisense defensin or defensin-like coding sequences.

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit ortargeting peptide (usually into vacuoles, vesicles, plastids and otherintracellular organelles) and signal peptide or sequences (usually tothe endoplasmic reticulum, Golgi apparatus, and outside of the cellularmembrane). By facilitating the transport of the protein intocompartments inside and outside the cell, these sequences may increasethe accumulation of gene products by protecting them from proteolyticdegradation. These sequences also allow for additional mRNA sequencesfrom highly expressed genes to be attached to the coding sequence of thegenes. Since mRNA being translated by ribosomes is more stable thannaked mRNA, the presence of translatable mRNA in front of the gene mayincrease the overall stability of the mRNA transcript from the gene andthereby increase synthesis of the gene product. Since transit and signalsequences are usually post-translationally removed from the initialtranslation product, the use of these sequences allows for the additionof extra translated sequences that may not appear on the finalpolypeptide. It further is contemplated that targeting of certainproteins may be desirable in order to enhance the stability of theprotein.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit or signal peptide willtransport the protein to a particular intracellular or extracellulardestination, respectively, and will then be post-translationallyremoved.

By employing a selectable or screenable marker, one can provide orenhance the ability to identify transformants. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker proteinand thus allow such transformed cells to be distinguished from cellsthat do not have the marker. Such genes may encode either a selectableor screenable marker, depending on whether the marker confers a traitwhich one can “select” for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by “screening” (e.g., the green fluorescent protein). Ofcourse, many examples of suitable marker proteins are known to the artand can be employed in the practice of the present disclosure.

Selectable marker transgenes may also be used with the presentdisclosure. As used herein the term “selectable marker transgene” refersto any transcribable DNA molecule whose expression in a transgenicplant, tissue or cell, or lack thereof, can be screened for or scored insome way. Selectable marker genes, and their associated selection andscreening techniques, for use in the practice of the present disclosureare known in the art and include, but are not limited to, transcribableDNA molecules encoding β-glucuronidase (GUS), green fluorescent protein(GFP), proteins that confer antibiotic resistance, and proteins thatconfer herbicide tolerance.

VII. Plant Cell Transformation Methods

Numerous methods for transforming chromosomes in a plant cell withrecombinant DNA are known in the art and are used in methods ofproducing a transgenic plant cell and plant. Two effective methods forsuch transformation are Agrobacterium-mediated transformation andmicroprojectile bombardment-mediated transformation. Microprojectilebombardment methods are illustrated in U.S. Pat. No. 5,015,580(soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880(corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208(corn); U.S. Pat. No. 6,399,861 (corn); U.S. Pat. No. 6,153,812 (wheat)and U.S. Pat. No. 6,365,807 (rice). Agrobacterium-mediatedtransformation methods are described in U.S. Pat. No. 5,159,135(cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,463,174(canola); U.S. Pat. No. 5,591,616 (corn); U.S. Pat. No. 5,846,797(cotton); U.S. Pat. No. 6,384,301 (soybean), U.S. Pat. No. 7,026,528(wheat) and U.S. Pat. No. 6,329,571 (rice), and US Patent ApplicationPublication Nos. US 2004/0087030 A1 (cotton), and US 2001/0042257 A1(sugar beet), all of which are incorporated herein by reference in theirentirety. Transformation of plant material is practiced in tissueculture on nutrient media, for example a mixture of nutrients that allowcells to grow in vitro. Recipient cell targets include, but are notlimited to, meristem cells, shoot tips, hypocotyls, calli, immature ormature embryos, and gametic cells such as microspores, pollen, sperm andegg cells. Callus can be initiated from tissue sources including, butnot limited to, immature or mature embryos, hypocotyls, seedling apicalmeristems, microspores and the like. Cells containing a transgenicnucleus are grown into transgenic plants.

In addition to direct transformation of a plant material with arecombinant DNA, a transgenic plant can be prepared by crossing a firstplant comprising a recombinant DNA with a second plant lacking therecombinant DNA. For example, recombinant DNA can be introduced into afirst plant line that is amenable to transformation, which can becrossed with a second plant line to introgress the recombinant DNA intothe second plant line. A transgenic plant with recombinant DNA providingan enhanced trait, for example, enhanced yield, can be crossed with atransgenic plant line having another recombinant DNA that confersanother trait, for example herbicide resistance or pest resistance orenhanced water use efficiency, to produce progeny plants havingrecombinant DNA that confers both traits. Typically, in such breedingfor combining traits the transgenic plant donating the additional traitis the male line and the transgenic plant carrying the base traits isthe female line. The progeny of this cross will segregate such that someof the plants will carry the DNA for both parental traits and some willcarry DNA for one parental trait; such plants can be identified bymarkers associated with parental recombinant DNA, for example, markeridentification by analysis for recombinant DNA or, in the case where aselectable marker is linked to the recombinant DNA, by application usinga selective agent such as a herbicide for use with a herbicide tolerancemarker, or by selection for the enhanced trait. Progeny plants carryingDNA for both parental traits can be crossed back into the female parentline multiple times, for example usually 6 to 8 generations, to producea progeny plant with substantially the same genotype as the originaltransgenic parental line but for the recombinant DNA of the othertransgenic parental line.

In transformation, DNA is typically introduced into only a smallpercentage of target plant cells in any one transformation experiment.Marker genes are used to provide an efficient system for identificationof those cells that are stably transformed by receiving and integratinga recombinant DNA molecule into their genomes. Preferred marker genesprovide selective markers which confer resistance to a selective agent,such as an antibiotic or an herbicide. Any of the herbicides to whichplants of this disclosure can be resistant is an agent for selectivemarkers. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells are those cells where,generally, the resistance-conferring gene is integrated and expressed atsufficient levels to permit cell survival. Cells can be tested furtherto confirm stable integration of the exogenous DNA. Commonly usedselective marker genes include those conferring resistance toantibiotics such as kanamycin and paromomycin (nptII), hygromycin B (aphIV), spectinomycin (aadA) and gentamycin (aac3 and aacC4) or resistanceto herbicides such as glufosinate (bar or pat), dicamba (DMO) andglyphosate (aroA or EPSPS). Examples of such selectable markers areillustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and6,118,047. Markers which provide an ability to visually screentransformants can also be employed, for example, a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

VIII. Transgenic Plants and Seeds

Transgenic plants derived from transgenic plant cells having atransgenic nucleus of this disclosure are grown to generate transgenicplants having an enhanced trait as compared to a control plant, andproduce transgenic seed and haploid pollen of this disclosure. Suchplants with enhanced traits are identified by selection of transformedplants or progeny seed for the enhanced trait. For efficiency aselection method is designed to evaluate multiple transgenic plants(events) comprising the recombinant DNA, for example multiple plantsfrom 2 to 20 or more transgenic events. Transgenic plants grown fromtransgenic seeds provided herein demonstrate improved agronomic traits,such as resistance to Fusarium stalk rot in maize.

Seed Treatment

In an aspect, a method, a corn plant, or a corn seed provided herein isused in combination with one or more pesticides including, but notlimited to, herbicides, fungicides (e.g., picoxystrobin, cyproconazole,tetraconazole, pyraclostrobin, metconazole, azoxystrobin, propiconazole,prothioconazole, trifloxystrobin), insecticides, microbiocides,nematicides, insect repellents, bactericides, and other substances usedto control pests. In another aspect, a method, a corn plant, or a cornseed provided herein is used in combination with one or more triazoles,strobilurins, acylamino acids, pyrimidines, pyridines, arylphenylketones, amides, benzanilides, imidazoles, dinitrophenols, morpholines,phenylsulfamides and organophosphorus cpds, derivatives thereof andcombinations thereof which can be applied as a seed treatment, a foliartreatment, a drench treatment, or a drip treatment.

In an aspect, corn seeds provided herein are untreated. In anotheraspect, corn seeds provided herein can be subjected to various andmultiple treatments. For example, without being limiting, the seeds canbe treated to improve germination by priming the seeds, by disinfectionto protect against seed borne pathogens, or both priming anddisinfection. In another example, seeds can be coated with any availablecoating to improve, for example, plantability, seed emergence, andprotection against seed borne pathogens. Seed coating can be any form ofseed coating including, but not limited to, pelleting, film coating, andencrustments.

IX. General Terms and Definitions

Systems, including automated systems for selecting plants that comprisea marker of interest and/or for correlating presence of the marker withresistance are also a feature of the invention. These systems caninclude probes relevant to marker locus detection, detectors fordetecting labels on the probes, appropriate fluid handling elements andtemperature controllers that mix probes and templates and/or amplifytemplates and systems instructions that correlate label detection to thepresence of a particular marker locus or allele.

In an aspect, this invention could be used on any plant. In anotheraspect, the plant is selected from the genus Zea. In another aspect, theplant is selected from the species Zea mays. In a further aspect, theplant is selected from the subspecies Zea mays L. ssp. mays. In anadditional aspect, the plant is selected from the group Zea mays L.subsp. mays Indentata, otherwise known as dent corn. In another aspect,the plant is selected from the group Zea mays L. subsp. mays Indurata,otherwise known as flint corn. In an aspect, the plant is selected fromthe group Zea mays L. subsp. mays Saccharata, otherwise known as sweetcorn. In another aspect, the plant is selected from the group Zea maysL. subsp. mays Amylacea, otherwise known as flour corn. In a furtheraspect, the plant is selected from the group Zea mays L. subsp. maysEverta, otherwise known as pop corn. Zea plants include hybrids,inbreds, partial inbreds, or members of defined or undefinedpopulations.

In a preferred aspect, the present invention provides a plant to beassayed for resistance or susceptibility to disease by any method todetermine whether a plant is resistant, susceptible, or whether itexhibits some degree of resistance or susceptibility. Populations ofplants can be similarly characterized in this manner, or furthercharacterized as segregating for the trait of disease resistance.

It is further understood that a plant of the present invention mayexhibit the characteristics of any relative maturity group. In anaspect, the maturity group is selected from the group consisting ofearly maturing varieties, mid-season maturing varieties, and full seasonvarieties.

The present invention also provides for parts of the plants of thepresent invention. Plant parts, without limitation, include seed,endosperm, ovule and pollen. In a particularly preferred aspect of thepresent invention, the plant part is a seed.

In another aspect, the corn seed can be subjected to various treatments.For example, the seeds can be treated to improve germination by primingthe seeds or by disinfection to protect against seed-borne pathogens. Inanother aspect, seeds can be coated with any available coating toimprove, for example, plantability, seed emergence, and protectionagainst seed-borne pathogens. Seed coating can be any form of seedcoating including, but not limited to, pelleting, film coating, andencrustments.

In another aspect, the corn plant can show a comparative resistancecompared to a non-resistant control corn plant. In this aspect, acontrol corn plant will preferably be genetically similar except for thedisease resistance allele or alleles in question. Such plants can begrown under similar conditions with equivalent or near equivalentexposure to the pathogen.

Various patent and non-patent publications are cited herein, thedisclosures of each of which are incorporated herein by reference intheir entireties.

As various modifications could be made in the constructions and methodsherein described and illustrated without departing from the scope of theinvention, it is intended that all matter contained in the foregoingdescription or shown in the accompanying drawings shall be interpretedas illustrative rather than limiting. The breadth and scope of thepresent invention should not be limited by any of the above-describedexemplary embodiments, but should be defined only in accordance with thefollowing claims appended hereto and their equivalents.

Descriptions of commonly used breeding terms and methods for crossingand producing hybrids that are used to describe present invention can befound in one of several reference books (Allard, “Principles of PlantBreeding,” John Wiley & Sons, NY, U. of CA, Davis, Calif., 50-98, 1960;Simmonds, “Principles of crop improvement,” Longman, Inc., NY, 369-399,1979; Sneep and Hendriksen, “Plant breeding perspectives,” Wageningen(ed), Center for Agricultural Publishing and Documentation, 1979; Fehr,In: Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph.,16:249, 1987; Fehr, “Principles of variety development,” Theory andTechnique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ.,Macmillan Pub. Co., NY, 360-376, 1987).

The definitions and methods provided define the present invention andguide those of ordinary skill in the art in the practice of the presentinvention. Unless otherwise noted, terms are to be understood accordingto conventional usage by those of ordinary skill in the relevant art.Examples of resources describing many of the terms related to molecularbiology used herein can be found in in Alberts et al., Molecular Biologyof The Cell, 5^(th) Edition, Garland Science Publishing, Inc.: New York,2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5thedition, Springer-Verlag: New York, 1991; King et al, A Dictionary ofGenetics, 6th ed, Oxford University Press: New York, 2002; and Lewin,Genes Icorn, Oxford University Press: New York, 2007. The nomenclaturefor DNA bases as set forth at 37 CFR § 1.822 is used.

Definitions

Terms defined herein have meanings as commonly understood by a person ofordinary skill in the areas relevant to the present invention. Termssuch as “a”, “an” and “the” are not intended to refer to only a singularentity, but include the general class of which a specific example may beused for illustration. The terminology herein is used to describespecific embodiments of the invention, but their usage does not delimitthe invention, except as outlined in the claims.

“Adjacent”, when used to describe a nucleic acid molecule thathybridizes to DNA containing a polymorphism, refers to a nucleic acidthat hybridizes to DNA sequences that directly about the polymorphicnucleotide base position. For example, a nucleic acid molecule that canbe used in a single base extension assay is “adjacent” to thepolymorphism.

“Allele” generally refers to an alternative nucleic acid sequence at aparticular locus; the length of an allele can be as small as 1nucleotide base, but is typically larger. For example, a first allelecan occur on one chromosome, while a second allele occurs on a secondhomologous chromosome, e.g., as occurs for different chromosomes of aheterozygous individual, or between different homozygous or heterozygousindividuals in a population. A favorable allele is the allele at aparticular locus that confers, or contributes to, an agronomicallydesirable phenotype, or alternatively, is an allele that allows theidentification of susceptible plants that can be removed from a breedingprogram or planting. A favorable allele of a marker is a marker allelethat segregates with the favorable phenotype, or alternatively,segregates with susceptible plant phenotype, therefore providing thebenefit of identifying disease prone plants. A favorable allelic form ofa chromosome interval is a chromosome interval that includes anucleotide sequence that contributes to superior agronomic performanceat one or more genetic loci physically located on the chromosomeinterval. “Allele frequency” refers to the frequency (proportion orpercentage) at which an allele is present at a locus within anindividual, within a line, or within a population of lines. For example,for an allele “A,” diploid individuals of genotype “AA,” “Aa,” or “aa”have allele frequencies of 1.0, 0.5, or 0.0, respectively. One canestimate the allele frequency within a line by averaging the allelefrequencies of a sample of individuals from that line. Similarly, onecan calculate the allele frequency within a population of lines byaveraging the allele frequencies of lines that make up the population.For a population with a finite number of individuals or lines, an allelefrequency can be expressed as a count of individuals or lines (or anyother specified grouping) containing the allele. An allele positivelycorrelates with a trait when it is linked to it and when presence of theallele is an indicator that the desired trait or trait form will occurin a plant comprising the allele. An allele negatively correlates with atrait when it is linked to it and when presence of the allele is anindicator that a desired trait or trait form will not occur in a plantcomprising the allele.

“Crossed” or “cross” means to produce progeny via fertilization (e.g.cells, seeds or plants) and includes crosses between plants (sexual) andself-fertilization (selfing).

“Elite line” means any line that has resulted from breeding andselection for superior agronomic performance. Numerous elite lines areavailable and known to those of skill in the art of corn breeding. An“elite population” is an assortment of elite individuals or lines thatcan be used to represent the state of the art in terms of agronomicallysuperior genotypes of a given crop species, such as corn. Similarly, an“elite germplasm” or elite strain of germplasm is an agronomicallysuperior germplasm, typically derived from and/or capable of giving riseto a plant with superior agronomic performance, such as an existing ornewly developed elite line of corn. In contrast, an “exotic line” or“exotic germplasm” is a line or germplasm derived from a plant notbelonging to an available elite line or strain of germplasm. In thecontext of a cross between two plants or lines of germplasm, an exoticgermplasm is not closely related by descent to the elite germplasm withwhich it is crossed. Most commonly, the exotic germplasm is not derivedfrom any known elite line of a crop, but rather is selected to introducegenetic elements (typically desired alleles) into a breeding program.

“Exogenous nucleic acid” is a nucleic acid that is not native to aspecified system (e.g., a germplasm, plant, variety, etc.), with respectto sequence, genomic position, or both. As used herein, the terms“exogenous” or “heterologous” as applied to polynucleotides orpolypeptides typically refers to molecules that have been artificiallysupplied to a biological system (e.g., a plant cell, a plant gene, aparticular plant species or variety or a plant chromosome under study)and are not native to that particular biological system. The terms canindicate that the relevant material originated from a source other thana naturally occurring source, or can refer to molecules having anon-natural configuration, genetic location or arrangement of parts. Incontrast, for example, a “native” or “endogenous” gene is a gene thatdoes not contain nucleic acid elements encoded by sources other than thechromosome or other genetic element on which it is normally found innature. An endogenous gene, transcript or polypeptide is encoded by itsnatural chromosomal locus, and not artificially supplied to the cell.

“Genetic element” or “gene” refers to a heritable sequence of DNA, i.e.,a genomic sequence, with functional significance. The term “gene” canalso be used to refer to, e.g., a cDNA and/or a mRNA encoded by agenomic sequence, as well as to that genomic sequence.

“Genotype” is the genetic constitution of an individual (or group ofindividuals) at one or more genetic loci, as contrasted with theobservable trait (the phenotype). Genotype is defined by the allele(s)of one or more known loci that the individual has inherited from itsparents. The term genotype can be used to refer to an individual'sgenetic constitution at a single locus, at multiple loci, or, moregenerally, the term genotype can be used to refer to an individual'sgenetic make-up for all the genes in its genome. A “haplotype” is thegenotype of an individual at a plurality of genetic loci. Typically, thegenetic loci described by a haplotype are physically and geneticallylinked, i.e., on the same chromosome interval. The terms “phenotype,” or“phenotypic trait” or “trait” refers to one or more trait of anorganism. The phenotype can be observable to the naked eye, or by anyother means of evaluation known in the art, e.g., microscopy,biochemical analysis, genomic analysis, an assay for a particulardisease resistance, etc. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, i.e., a “single genetrait.” In other cases, a phenotype is the result of several genes.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells that can be cultured into a whole plant.

“Linkage disequilibrium” or “LD” refers to a non-random segregation ofgenetic loci or traits (or both). In either case, linkage disequilibriumimplies that the relevant loci are within sufficient physical proximityalong a length of a chromosome so that they segregate together withgreater than random (i.e., non-random) frequency (in the case ofco-segregating traits, the loci that underlie the traits are insufficient proximity to each other) Linked loci co-segregate more than50% of the time, e.g., from about 51% to about 100% of the time. Theterm “physically linked” is sometimes used to indicate that two loci,e.g., two marker loci, are physically present on the same chromosome.Advantageously, the two linked loci are located in close proximity suchthat recombination between homologous chromosome pairs does not occurbetween the two loci during meiosis with high frequency, e.g., such thatlinked loci cosegregate at least about 90% of the time, e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time.

“Locus” a chromosome region where a polymorphic nucleic acid, traitdeterminant, gene or marker is located. The loci of this inventioncomprise one or more polymorphisms in a population; i.e., alternativealleles are present in some individuals. A “gene locus” is a specificchromosome location in the genome of a species where a specific gene canbe found.

“Marker Assay” means a method for detecting a polymorphism at aparticular locus using a particular method, e.g. measurement of at leastone phenotype (such as seed color, flower color, or other visuallydetectable trait), restriction fragment length polymorphism (RFLP),single base extension, electrophoresis, sequence alignment, allelicspecific oligonucleotide hybridization (ASO), random amplifiedpolymorphic DNA (RAPD), microarray-based technologies, and nucleic acidsequencing technologies, etc. “Marker Assisted Selection” (MAS) is aprocess by which phenotypes are selected based on marker genotypes.

“Molecular phenotype” is a phenotype detectable at the level of apopulation of one or more molecules. Such molecules can be nucleicacids, proteins, or metabolites. A molecular phenotype could be anexpression profile for one or more gene products, e.g., at a specificstage of plant development, in response to an environmental condition orstress, etc.

“Operably linked” refers to the association of two or more nucleic acidelements in a recombinant DNA construct, e.g. as when a promoter isoperably linked with DNA that is transcribed to RNA whether forexpressing or suppressing a protein. Recombinant DNA constructs can bedesigned to express a protein which can be an endogenous protein, anexogenous homologue of an endogenous protein or an exogenous proteinwith no native homologue. Alternatively, recombinant DNA constructs canbe designed to suppress the level of an endogenous protein, e.g. bysuppression of the native gene. Such gene suppression can be effectivelyemployed through a native RNA interference (RNAi) mechanism in whichrecombinant DNA comprises both sense and anti-sense oriented DNA matchedto the gene targeted for suppression where the recombinant DNA istranscribed into RNA that can form a double-strand to initiate an RNAimechanism. Gene suppression can also be effected by recombinant DNA thatcomprises anti-sense oriented DNA matched to the gene targeted forsuppression. Gene suppression can also be effected by recombinant DNAthat comprises DNA that is transcribed to a microRNA matched to the genetargeted for suppression.

“Percent identity” or “% identity” means the extent to which twooptimally aligned DNA or protein segments are invariant throughout awindow of alignment of components, for example nucleotide sequence oramino acid sequence. An “identity fraction” for aligned segments of atest sequence and a reference sequence is the number of identicalcomponents that are shared by sequences of the two aligned segmentsdivided by the total number of sequence components in the referencesegment over a window of alignment which is the smaller of the full testsequence or the full reference sequence.

“Phenotype” means the detectable characteristics of a cell or organismwhich can be influenced by genotype.

“Plant” refers to a whole plant any part thereof, or a cell or tissueculture derived from a plant, comprising any of: whole plants, plantcomponents or organs (e.g., leaves, stems, roots, etc.), plant tissues,seeds, plant cells, and/or progeny of the same. A plant cell is abiological cell of a plant, taken from a plant or derived throughculture from a cell taken from a plant.

“Polymorphism” means the presence of one or more variations in apopulation. A polymorphism may manifest as a variation in the nucleotidesequence of a nucleic acid or as a variation in the amino acid sequenceof a protein. Polymorphisms include the presence of one or morevariations of a nucleic acid sequence or nucleic acid feature at one ormore loci in a population of one or more individuals. The variation maycomprise but is not limited to one or more nucleotide base changes, theinsertion of one or more nucleotides or the deletion of one or morenucleotides. A polymorphism may arise from random processes in nucleicacid replication, through mutagenesis, as a result of mobile genomicelements, from copy number variation and during the process of meiosis,such as unequal crossing over, genome duplication and chromosome breaksand fusions. The variation can be commonly found or may exist at lowfrequency within a population, the former having greater utility ingeneral plant breeding and the latter may be associated with rare butimportant phenotypic variation. Useful polymorphisms may include singlenucleotide polymorphisms (SNPs), insertions or deletions in DNA sequence(Indels), simple sequence repeats of DNA sequence (SSRs), a restrictionfragment length polymorphism, and a tag SNP. A genetic marker, a gene, aDNA-derived sequence, a RNA-derived sequence, a promoter, a 5′untranslated region of a gene, a 3′ untranslated region of a gene,microRNA, siRNA, a resistance locus, a satellite marker, a transgene,mRNA, ds mRNA, a transcriptional profile, and a methylation pattern mayalso comprise polymorphisms. In addition, the presence, absence, orvariation in copy number of the preceding may comprise polymorphisms.

A “population of plants” or “plant population” means a set comprisingany number, including one, of individuals, objects, or data from whichsamples are taken for evaluation, e.g. estimating QTL effects. Mostcommonly, the terms relate to a breeding population of plants from whichmembers are selected and crossed to produce progeny in a breedingprogram. A population of plants can include the progeny of a singlebreeding cross or a plurality of breeding crosses, and can be eitheractual plants or plant derived material, or in silico representations ofthe plants. The population members need not be identical to thepopulation members selected for use in subsequent cycles of analyses orthose ultimately selected to obtain final progeny plants. Often, a plantpopulation is derived from a single biparental cross, but may alsoderive from two or more crosses between the same or different parents.Although a population of plants may comprise any number of individuals,those of skill in the art will recognize that plant breeders commonlyuse population sizes ranging from one or two hundred individuals toseveral thousand, and that the highest performing 5-20% of a populationis what is commonly selected to be used in subsequent crosses in orderto improve the performance of subsequent generations of the population.

“Resistance” or “improved resistance” in a plant to disease conditionsis an indication that the plant is more able to reduce disease burdenthan a non-resistant or less resistant plant. Resistance is a relativeterm, indicating that a “resistant” plant is more able to reduce diseaseburden compared to a different (less resistant) plant (e.g., a differentcorn line) grown in similar disease conditions. One of skill willappreciate that plant resistance to disease conditions varies widely,and can represent a spectrum of more-resistant or less-resistantphenotypes. However, by simple observation, one of skill can generallydetermine the relative resistance of different plants, plant lines, orplant families under disease conditions, and furthermore, will alsorecognize the phenotypic gradations of “resistant.”

“Resistance locus” means a locus that contributes resistance, tolerance,or susceptibility to Fusarium stalk rot.

“Resistance allele” means the nucleic acid sequence associated withresistance or tolerance to disease.

“Recombinant” in reference to a nucleic acid or polypeptide indicatesthat the material (e.g., a recombinant nucleic acid, gene,polynucleotide, polypeptide, etc.) has been altered by humanintervention. The term recombinant can also refer to an organism thatharbors recombinant material, e.g., a plant that comprises a recombinantnucleic acid is considered a recombinant plant.

“Tolerance” or “improved tolerance” in a plant to disease conditions isan indication that the plant is less affected by disease conditions withrespect to yield, survivability and/or other relevant agronomicmeasures, compared to a less resistant, more “susceptible” plant.Tolerance is a relative term, indicating that a “tolerant” plantsurvives and/or produces better yields in disease conditions compared toa different (less tolerant) plant (e.g., a different corn line strain)grown in similar disease conditions. One of skill will appreciate thatplant tolerance to disease conditions varies widely, and can represent aspectrum of more-tolerant or less-tolerant phenotypes. However, bysimple observation, one of skill can generally determine the relativetolerance or susceptibility of different plants, plant lines or plantfamilies under disease conditions, and furthermore, will also recognizethe phenotypic gradations of “tolerant.”

“Transgenic plant” refers to a plant that comprises within its cells aheterologous polynucleotide. Generally, the heterologous polynucleotideis stably integrated within the genome such that the polynucleotide ispassed on to successive generations. The heterologous polynucleotide maybe integrated into the genome alone or as part of a recombinantexpression cassette. “Transgenic” is used herein to refer to any cell,cell line, callus, tissue, plant part or plant, the genotype of whichhas been altered by the presence of heterologous nucleic acid includingthose transgenic organisms or cells initially so altered, as well asthose created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextrachromosomal) by conventional plant breeding methods (e.g., crosses)or by naturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

“Vector” is a polynucleotide or other molecule that transfers nucleicacids between cells. Vectors are often derived from plasmids,bacteriophages, or viruses and optionally comprise parts which mediatevector maintenance and enable its intended use. A “cloning vector” or“shuttle vector” or “sub cloning vector” contains operably linked partsthat facilitate subcloning steps (e.g., a multiple cloning sitecontaining multiple restriction endonuclease sites). The term“expression vector” as used herein refers to a vector comprisingoperably linked polynucleotide sequences that facilitate expression of acoding sequence in a particular host organism (e.g., a bacterialexpression vector or a plant expression vector).

“Yield” is the culmination of all agronomic traits as determined by theproductivity per unit area of a particular plant product of commercialvalue. “Agronomic traits,” include the underlying genetic elements of agiven plant variety that contribute to yield over the course of growingseason.

EXAMPLES Example 1. Identification of FSR QTLs

Corn plants were inoculated 10 days after flowering by injecting aninternode of each stalk with 2×10⁵ Fusarium moniliforme spores suspendedin 2 mL of distilled water. Six weeks after inoculation, the severity ofFusarium stalk rot in each plant was visually assessed by splittingstalks longitudinally to expose the pith. Internodes at the site ofinoculation were examined to determine the percent of the internodeshowing visual lesions characteristic of the disease (necrosis), assummarized in Table 1. The individual plant scores of each row were thenaveraged to generate a final score for the row.

TABLE 1 Rating scale of relative FSR infection resistance phenotypes. %Internode Necrosis Score Rating  0-10% 1 Highly resistant 10-20% 2Highly resistant 20-30% 3 Resistant 30-40% 4 Resistant 40-50% 5Intermediate 50-60% 6 Susceptible 60-70% 7 Susceptible   >80% 8 Highlysusceptible >80% and necrosis 9 Highly susceptible expands to adjacentinternode

Parental lines were selected from Monsanto proprietary inbred lines asshown in Table 2.

TABLE 2 Bi-parental mapping populations. Mapping Resistant SusceptiblePopulation Population Population Cross Line Line Type Size ACV119351/CV120707 CV119351 CV120707 Double-haploid 192 BCV122159/CV119964 CV119964 CV122159 F3 204 C CV335662*2/CV353184CV353184 CV335662 BC1F4 192 D CV123496/CV124239 CV123496 CV124239Double-haploid 208 E CV355838/CV123302 CV355838 CV123302 Double-haploid108

Plants from all mapping populations were then genotyped using SNPmarkers that collectively spanned each chromosome in the maize genome.The primer sequences for amplifying exemplary SNP marker loci linked tothe FSR and the probes used to genotype the corresponding SNP sequencesare provided in Table 3. One of skill in the art will recognize thatsequences to either side of the given primers can be used in place ofthe given primers, so long as the primers can amplify a region thatincludes the allele to be detected. The precise probe used for detectioncan vary, e.g., any probe that can identify the region of a markeramplicon to be detected can be substituted for those probes exemplifiedherein. Configuration of the amplification primers and detection probescan also be varied. Thus, the invention is not limited to the primers,probes, or marker sequences specifically recited herein.

TABLE 3 Exemplary SNP markers associated with FSR resistance. SEQ ID NO.SEQ SNP Fwd Rev ID NO. Position Primer Primer Probe 1 Probe 2 1 351 2855 82 109 2 338 29 56 83 110 3 341 30 57 84 111 4 329 31 58 85 112 5 27532 59 86 113 6 323 33 60 87 114 7 918 34 61 88 115 8 262 35 62 89 116 9118 36 63 90 117 10 115 37 64 91 118 11 209 38 65 92 119 12 262 39 66 93120 13 101 40 67 94 121 14 82 41 68 95 122 15 618 42 69 96 123 16 101 4370 97 124 17 117 44 71 98 125 18 101 45 72 99 126 19 101 46 73 100 12720 279 47 74 101 128 21 462 48 75 102 129 22 27 49 76 103 130 23 148 5077 104 131 24 204 51 78 105 132 25 342 52 79 106 133 26 133 53 80 107134 27 456 54 81 108 135

In an illustrative example, SNP marker SEQ ID NO: 1 can be amplifiedusing the primers described in Table 3 as SEQ ID NO: 28 (forward primer)and SEQ ID NO: 55 (reverse primer), and detected with probes indicatedas SEQ ID NO: 82 (Probe 1) and SEQ ID NO: 109 (Probe 2).

Marker-trait association studies were performed using both single-markeranalysis (SMA) and composite interval mapping (CIM). For each marker,the thresholds of likelihood ratio between full and null models for CIMwere based on 1000 random permutation tests and the thresholds (p-value)for SMA were based on 10,000 random permutation tests (Churchill andDoerg, 1994). The composite interval mapping (CIM) analysis revealedseveral strong QTLs associated with FSR resistance. Genetic map loci arerepresented in cM, with position zero being the first (most distal)marker known at the beginning of the chromosome on Monsanto's internalconsensus genetic map. Each row of Table 4 provides mapping populationID, number of SNP markers genotyped, resistant parent, chromosomeposition, the peak of the Likelihood ratio corresponding to FSRresistance, QTL interval where left and right flanking positions areshown, p-value, additive effect, and the phenotypic variance (R²) ofindividual QTL.

TABLE 4 CIM results from all mapping populations. QTL Positions (cM)Mapping Resistant Left Right Population #Mk Parent Chr Peak Flank Flankp-value Additive QTL R² A 292 CV119351 2 175.6 173.6 186.1 0.01 0.760.149 B 128 CV119964 5 144.8 138.2 154.8 0.01 0.82 0.18 C 169 CV353184 654.7 44.5 62.7 0.01 0.69 0.28 D 137 CV123496 6 56.9 48.9 67.8 0.01 0.480.18 E 149 CV355838 7 60.3 47.3 64.5 0.01 0.55 0.18 *p-value is based on1,000 permutation tests

The identified QTLs were designated as FSR_2.01, FSR_5.01, FSR_6.01 andFSR_7.01 (Table 5).

TABLE 5 Summary of FSR QTLs QTL interval IBM2008 Map Flanking MarkersQTL Chromosome (cM) (IcM) Left Right Designation 2 173-187 568-607npi610 gpm365a FSR_2.01 5 138-155 483-524 hmg2 agrp90 FSR_5.01 6 44-68216-318 gpm674c TIDP5534 FSR_6.01 7 47-65  99-248 TIDP2919 IDP5002FSR_7.01 cM = centiMorgans; IcM = map units of the IBM2 2008 NeighborsGenetic Map; flanking markers available on the publically available IBM22008 Neighbors Genetic Map

In Table 5, “IcM” refers to the map units of the IBM2 2008 NeighborsGenetic Map, which was generated with an intermated recombinant inbredpopulation (syn 4) that resulted in approximately a four-fold increasein the number of meiosies as compared to the typical recombinationexperiment that is used to generate centiMorgan (cM) distances (Lee, etal., 2002, Plant Mol Biol 48:453 and the Maize Genetics and GenomicsDatabase). “cM” refers to the classical definition of a centimorganwherein one cM is equal to a 1% chance that a trait at one genetic locuswill be separated from a trait at another locus due to crossing over ina single generation (meaning the traits cosegregate 99% of the timeduring meiosis), and this definition is used herein to delineate maplocations pertaining to this invention.

Table 6 lists the effect estimates on FSR rating score for each marker(SEQ ID NO.) linked to FSR resistance based on SMA. Each row of Table 6provides the SEQ ID NO. of the marker, chromosome position, markerposition on Monsanto's internal consensus genetic map and the Neighbors2008 maize genomic map (publicly available on the internet; Andorf, etal. (2015) MaizeGDB 2015: New tools, data, and interface for the maizemodel organism database. Nucleic Acids Research), genetic source ofresistant allele, resistant allele, susceptible allele, the estimatedeffect that the marker polymorphism had on the FSR rating score andp-value based on permutation test. For example, SEQ ID NO: 14 wasassociated with a reduction of 0.51 in FSR rating score by one copy ofthe resistant allele. However, one of skill in the art will recognizethat a “resistant” allele at one locus may be a “susceptible” allele ina different genetic background. Thus, the invention is not limited tothe “resistant” and “susceptible” alleles exemplified herein.

TABLE 6 Estimate effects of markers linked to FSR resistance from allmapping populations by SMA. Genetic SEQ MON Source of Single PermutationID Map IBM2008 Resistant Resistant Susceptible Allele Testing NO.Chromosome cM Map IcM Allele allele allele Effect Probability 1 2 170.6558.6 CV119351 C T 0.255 0.001 2 2 176.9 582.4 CV119351 C T 0.568 0.0013 2 186.1 603.2 CV119351 A C 0.405 0.001 4 5 129.7 453.9 CV119964 C G0.473 0.001 5 5 132.8 465.5 CV119964 A G 0.512 0.001 6 5 148.8 508.5CV119964 G A 0.703 0.001 7 5 157.7 532.5 CV119964 G A 0.566 0.001 8 638.6 201.1 CV353184 A G 0.445 0.001 9 6 50.7 234.3 CV353184 T C 0.6010.001 10 6 59.4 267.1 CV353184 G A 0.586 0.001 11 6 68.3 318.7 CV353184G C 0.445 0.001 12 6 38.6 201.1 CV123496 G A 0.364 0.001 13 6 48.9 229.4CV123496 A G 0.421 0.001 14 6 52.7 0.0 CV123496 C T 0.51 0.001 15 6 58.6262.3 CV123496 G C 0.447 0.001 16 6 62.6 295.7 CV123496 C T 0.422 0.00117 6 63.6 300.8 CV123496 T C 0.376 0.001 18 6 64.1 302.7 CV123496 C T0.441 0.001 19 6 64.5 304.1 CV123496 G T 0.384 0.001 20 6 66.0 312.3CV123496 A G 0.379 0.001 21 6 66.3 313.2 CV123496 C T 0.36 0.001 22 667.4 316.2 CV123496 G A 0.361 0.001 23 6 69.0 320.7 CV123496 A G 0.3880.001 24 7 45.3 95.5 CV355838 G C 0.484 0.001 25 7 51.8 118.5 CV355838 CT 0.426 0.001 26 7 59.3 152.1 CV355838 G A 0.432 0.001 27 7 65.7 248.0CV355838 A T 0.522 0.001 *p-value is based on 10,000 permutation tests

Example 2. Validation of FSR QTLs

Plants with or without resistant FSR QTL were derived. Plants carryingthe resistant allele of FSR-2.01 or FSR-6.01 showed significantreductions in FSR rating score when compared to plants carrying thesusceptible allele (Table 7).

TABLE 7 Validation of FSR QTLs QTL Resistance FSR Infection interval QTLProfile Score StdErr p-value FSR_2.01 Absent 2.97 0.16 2.22E−02 Present2.6 0.17 FSR_6.01 Absent 3.5 0.1 2.05E−07 Present 2.8 0.1 Present: withresistant QTL Absent: without resistant QTL

Example 3. Fine-Mapping of FSR Resistance QTL on Chromosome 6

In order to obtain additional recombinants, CV353184/CV056579 derived F1lines were selected, self-crossed, and harvested. CV353184 is a FSRresistant line. CV056579 is a FSR susceptible line and is disclosed inU.S. Patent Publication No. 2016/0270355, published on Sep. 22, 2016,the entire content of which is incorporated by reference herein. Theresulting segregating F2 kernels were chipped and genotyped with 23 SNPmarkers within 46.9-64.2 cM on chromosome 6 based on the internalconsensus genetic map (Table 8).

TABLE 8 Primers and probes used for detecting SNPs linked to FSR-6.01 inthe fine-mapping SEQ ID NO. SEQ SNP Fwd Rev ID NO. Position PrimerPrimer Probe 1 Probe 2 136 101 155 174 193 212 13 101 40 67 94 121 137101 156 175 194 213 138 162 157 176 195 214 9 118 36 63 90 117 139 101158 177 196 215 140 74 159 178 197 216 141 130 160 179 198 217 142 409161 180 199 218 143 101 162 181 200 219 144 101 163 182 201 220 145 101164 183 202 221 10 115 37 64 91 118 146 101 165 184 203 222 147 102 166185 204 223 148 101 167 186 205 224 16 101 43 70 97 124 149 179 168 187206 225 150 747 169 188 207 226 151 101 170 189 208 227 152 101 171 190209 228 153 101 172 191 210 229 154 101 173 192 211 230

Kernels were bulked based on their haplotypes within this QTL region.These bulks (10-20 plants/bulk) were then planted and subsequentlyscreened for FSR resistance via progeny test at three locations. InTable 9, grey cells with bold and underlined font indicated that thebulk carried the same alleles as the susceptible inbred line at thespecific SNP position. White cells Cells without bold or underlined fontindicated that the bulk carried the same allele as the resistant inbredline at the specific SNP position. For example, bulk “k” shared the samecandidate QTL region as the susceptible inbred line, CV056579. Theindividual plant scores of each bulk were averaged. The average wasreported as a final score for the bulk and then compared with that ofsusceptible line. Bulk “d”, “e” and “f” displayed significantly reducedFSR severity (highlighted by black box, p-value ≤0.05). Based on thecommon SNP (highlighted by black oval) of these bulks, SEQ ID NO: 142was identified as the peak SNP marker. The two closest SNP markersflanking SEQ ID NO: 142 are SEQ ID NO: 141 and SEQ ID NO: 143.

For example, bulk “a” shared the same alleles as the resistant inbredline (CV353184) at the SNP positions represented by SEQ ID NO: 136 andSEQ ID NO: 13 (highlighted by white cells without bold or underlinedfont). Bulk “a” shared the same alleles as the susceptible inbred line(CV056579) at the rest of SNP positions (highlighted by grey cells withbold and underlined font).

The QTL region associated with FSR resistance was fine-mapped to53.9-56.8 cM on chromosome 6 based on the internally-derived genetic map(Table 10).

TABLE 10 Summary of fine-mapping result SEQ MON IBM2008 ID NO. Map cMMarker Profile Map IcM 141 53.9 Left flanking marker 242.7 142 54.1 QTLpeak 243.2 143 56.8 Right flanking marker 255.2

1. A method of obtaining a corn plant with enhanced Fusarium stalk rotresistance comprising: a) providing a population of corn plants; b)detecting in said plants the presence of a Fusarium stalk rot resistanceallele at a polymorphic locus genetically linked to a chromosomalsegment flanked by marker loci TIDP2919 and IDP5002 on chromosome 7; andc) selecting from said population at least a first plant comprising saidallele and enhanced Fusarium stalk rot resistance compared to a plantlacking said allele. 2.-5. (canceled)
 6. The method of claim 1, whereinsaid segment is flanked by marker loci SEQ ID NO: 24 and SEQ ID NO: 27on chromosome
 7. 7. The method of claim 1, wherein said polymorphiclocus comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 24-27.
 8. The method of claim 1, furtherdefined as comprising selecting from said population at least twoplants, thereby forming a population of corn plants comprising saidallele and enhanced Fusarium stalk rot resistance compared to a plantlacking said allele.
 9. The method of claim 1, wherein said Fusariumstalk rot resistance allele was introgressed into said population ofcorn plants from a starting plant or population of corn plantscontaining said allele.
 10. The method of claim 1, further comprisingproducing a progeny plant with Fusarium stalk rot resistance from saidfirst plant.
 11. The method of claim 10, wherein producing the progenyplant comprises marker-assisted selection for Fusarium stalk rotresistance.
 12. The method of claim 10, wherein the progeny plant is anF2-F6 progeny plant.
 13. The method of claim 10, wherein producing theprogeny plant comprises backcrossing.
 14. A method of producing a cornplant with enhanced Fusarium stalk rot resistance comprising: a)crossing a first corn plant comprising a Fusarium stalk rot resistanceallele with a second corn plant of a different genotype to produce oneor more progeny plants; and b) selecting a progeny plant based on thepresence of said allele at a polymorphic locus genetically linked to achromosomal segment flanked by marker loci TIDP2919 and IDP5002 onchromosome 7; wherein said allele confers enhanced resistance toFusarium stalk rot compared to a plant lacking said allele. 15.-18.(canceled)
 19. The method of claim 14, wherein said segment is flankedby marker loci SEQ ID NO: 24 and SEQ ID NO: 27 on chromosome
 7. 20. Themethod of claim 14, wherein said polymorphic locus comprises a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 24-27.21. The method of claim 14, wherein the progeny plant is an F2-F6progeny plant.
 22. The method of claim 14, wherein producing the progenyplant comprises backcrossing.
 23. The method of claim 22, whereinbackcrossing comprises from 2-7 generations of backcrosses.
 24. Themethod of claim 22, wherein backcrossing comprises marker-assistedselection in at least two generations.
 25. The method of claim 24,wherein backcrossing comprises marker-assisted selection in allgenerations.
 26. The method of claim 14, wherein the first corn plant isan inbred or a hybrid.
 27. The method of claim 14, wherein the secondcorn plant is an agronomically elite corn plant.
 28. The method of claim27, wherein the agronomically elite corn plant is an inbred or a hybrid.